Cellular arrays for the identification of altered gene expression

ABSTRACT

The present invention relates to the generation and use of a cellular array or a cellular array in combination with other genome-registered arrays (an array of arrays) for the determination of gene function and/or perturbation mode of action. Each cellular array consists of a number of microbial strains. Each strain comprises one reporter gene fusion made up of a gene or gene fragment operably linked to a reporter gene. Each gene or gene fragment has been “registered” or mapped to a specific location in the genome of the organism. The genome-registered collection of the invention may be used to determine alterations in gene expression under a variety of conditions. Such collections are amenable to rapid assay and may be used to confirm, correct or augment data generated from DNA micro array technology

[0001] This application claims the benefit of U.S. ProvisionalApplication No. 60/197,348 filed Apr. 14, 2000.

FIELD OF THE INVENTION

[0002] This invention is in the field of bacterial gene expression. Morespecifically, this invention describes a method to monitortranscriptional changes on a genome-wide scale using a genome-registeredgene fusion collection.

BACKGROUND OF THE INVENTION

[0003] DNA array analysis is a powerful method for comprehensive genomeanalysis of gene expression. Currently, this approach is the onlyavailable method for massively parallel analyses that allow theexpression of each gene of a bacterial genome to be characterizedsimultaneously (Richmond et al., (1999) Nucleic Acids Res.27:3821-3835., 17, 25; Tao et al., (1999), J. Bacteriol. 181:6425-6440;Wilson et al., (1999) Proc. Natl. Acad. Sci. U.S.A. 96:12833-12838).

[0004] Richmond et al. ((1999) Nucleic Acids Research, 27:3821-3835) hasrecently reported genome-wide expression profiling of E. coli at thesingle ORF level of resolution. Changes in RNA levels after exposure toheat shock or IPTG were analyzed using comprehensive low density blotsof individual ORFs on a nylon matrix and comprehensive high densityarrays of individual ORFs spotted on glass slides. The results of thetwo methods were compared. Richmond et al. states that radioactiveprobe/spot blots are inferior to fluorescent probe/micro-arrays.Moreover, the comparison of heat shock treatment between the two methodsis fundamentally flawed since the RNA analyzed with spot blots werederived from broth grown cultures while those analyzed with micro-arrayswere derived from cells grown in defined media. Despite the power ofthis new methodology, there are several problems that limit thereliability of results. For example, artifacts may arise during theisolation of microbial RNA (Tao et al., (1999) J. Bacteriol.181:6425-6440) or from cross hybridization to paralogous genes (Richmondet al., (1999) Nucleic Acids Res. 27:3821-3835, 17, 25).

[0005] Another limitation of DNA array methodology is that RNA must beisolated, converted into DNA by reverse transcriptase with concomitantincorporation of fluorescent labels. These steps make it unlikely thatfacile high throughput screens could be developed based on DNA arraytechnology. Thus, there exists a need for a method that adapts resultsfrom DNA array technology into high throughput screens. For the reasonsmentioned above and others, alternative genome-wide expression profilingmethod as well as rapid methods to independently verify results from DNAarray experiments are needed.

[0006] Gene fusion technology is an established method for geneexpression monitoring. For example, the initial discovery of the SOS(DNA damage responsive) regulon of E. coli was done by Kenyon and Walker((1980) Proc. Natl. Acad. Sci. U.S.A. 77:2819-2823) by comparing thetranscriptional responses of Escherichia coli to mitomycin C (MMC), aDNA damaging agent that intercalates into and forms a covalentattachment with double-stranded DNA. While these early experimentsattempted to scan the bulk of the E. coli genome by using a transposonthat put the lacZYA operon under the control of many promoter regions,it was not known if the entire genome had been surveyed because of therandom nature of transposition and unknown location of the majority oftransposition events. Accordingly, additional SOS regulon genes havebeen identified since these early experiments (Lomba et al., (1997)Microbiol Lett 156:119-122; Walker, (1996) In Escherichia coli andSalmonella: Cellular and Molecular Biology. ASM Press, pp 1400 1416).

[0007] LaRossa et al. (U.S. Pat. No. 5,683,868) has transformed E. coliwith at construct comprised of luxCDABE operably linked to a variety ofstress promoters. They have used the microorganisms to detect a varietyof environmental insults such as Ethanol, CdCl₂ and toluene. Thepresence of sublethal concentration of insults is indicated by anincrease in bioluminescence. However, in order to generate thetransformed host, the stress promoters has to be identified andcharacterized. Furthermore, this method is limited to the stressresponse only.

[0008] Ashby and Rine (U.S. Pat. No. 5,569,588) reported a method tomeasure the transcriptional responsiveness of an organism to a candidatedrug by detecting reporter gene product signals from separately isolatedcells of a target organism on genome-wide bases. Each cell contains arecombinant construct with the reporter gene operatively linked to adifferent endogenous transcriptional regulatory element of the targetorganism When cells were treated with a candidate drug, thetranscriptional responsiveness of the organism to the candidate drug wasmeasured by the detecting the reporter signal from each cells. However,this method is useful with the organism only after the majority oftranscriptional regulatory elements of the target organism are known andmapped. Furthermore, the reporter signals are measured only after cellsreached homeostasis in the presence of drug. The initial transcriptionalresponses to chemicals are not considered.

[0009] The Lux-A Collection of random E. coli genomic DNA fused to theluxCDABE had been used to screen for those gene fusions for whichexpression was induced by treatment with the herbicide sulfometuronmethyl. The DNA sequence of 19 of these sulfometuron methyl induciblegene fusions (smi-lux) was determined and used to identify the promotercontrolling expression of the luxCDABE reporter (Van Dyk et al., (1998)J. Bacteriol. 180:785-792); the remaining 8047 gene fusions remainedunidentified.

[0010] LaRossa and Van Dyk (U.S. Pat. No. 6,025,131) developed a methodfor the identification of gene regulatory regions, responsive to aparticular cellular stress, such as that produced by herbicides or cropprotection chemicals by randomly fusing regulatory regions to abacterial luminescent gene complex where contacting the fusion in asuitable host with a cellular insult producing a cellular stress resultsin detection of that cellular stress by an increase in cellularluminescence. However, this method was limited to the perturbations inliquid media; luminescent responses were not detected on solid mediumfollowing overnight growth in the presence of a chemical stress.Furthermore, it did not allow regulatory region activity analysis ingenome-wide scale.

[0011] The problem to be solved therefore is to provide a way to measureand follow the changes in gene expression using a genome-registeredcollection of reporter gene fusions in a manner that allows detection ofinitial transcriptional responses, and provide a way to cross-validatethe results from other method (i.e., microarray) as well as to determinepromoter and operon structure of genes, and further provide a way totest cellular responses to various environmental and genetic changes inhigh throughput manner.

SUMMARY OF THE INVENTION

[0012] A new method for the use of genome registered collection ofreporter gene fusion is disclosed. Fragments of genomic DNA of hostorganism were fused to promoterless reporter gene. The reporter genefusions were generated using restriction enzyme digestion, physicalshearing of the genomic DNA, PCR, and transposition techniques. Thereporter gene complexes were genome registered against the host genomeon the basis of homology. Gene expression of each reporter gene complexis measure as reporter gene activity. The present invention provides ameans to measure the changes in gene expression profiles in genome widescale under various conditions in high throughput manner. In addition tobeing a stand-alone high throughput method, the present invention alsoprovides a way to validate other genome-wide assays such as DNAmicroarray. The present invention also provides a method to confirm theresponse of several promoters to a particular insult (a condition orchemical of interest) as well as to identify a number of previouslyunknown operons responsive to that insult. Comparison of the geneexpression patterns of two samples differing in one variable is alsopossible using this method. The present invention also provides themethod to use an array of arrays by generating gene expression profilesthat yield information relevant to understanding gene function and modesof chemical action. Such information can be gained by analysis ofgenetic alterations resulting in loss of function, reduced levels ofgene products, or over-expression of gene products. Thus, an array ofarrays can be used to enhance both mode of action studies and functionalgenomics.

[0013] In this invention, the sequencing and genome-registering of themajority of the Lux-A Collection members were completed. Lux fusions inthe Lux-A Collection were fragments of E. coli genomic DNA fused topromoterless luxCDABE gene. The genome-registered collection of luxfusions were examined for the biological responses measured by changesin bioluminescence. The present invention provides a means to measurethe changes in gene expression profiles under various conditions.

[0014] In addition to being a stand-alone high throughput method, thepresent invention also provides the way to validate or detectfalse-positive result from other genome wide assays such as microarray.

[0015] The present invention provides a method to confirm the responseof several promoters to a particular insult as well as to identify anumber of previously unknown operons responsive to that insult.

[0016] The present invention provides a method for comparing the geneexpression patterns of two samples differing in one variable. Thevariables may include but not limited to genotype, media, temperature,depletion or addition of nutrient, addition of an inhibitor, physicalassault, biological assaults, irradiation, heat, cold, elevated orlowered pressure, desiccation, low or high ionic strength, and growthphases.

[0017] The present invention also provides the method to use an “arrayof arrays” by generating gene expression profiles that yield informationrelevant to understanding gene function and modes of chemical action.Such information can be gained by analysis of genetic alterationsresulting in loss of function, reduced levels of gene products, orover-expression of gene products. Thus, an array of arrays can be usedto enhance both modes of action studies and functional genomics.

[0018] Thus the invention provides a method for identifying altered geneexpression between at least two genome-registered collectionscomprising:

[0019] (a) assembling at least two genome-wide scale, genome-registeredcollections;

[0020] (b) perturbing each collection from (a) with at least oneperturbation;

[0021] (c) measuring the response of each collection to eachperturbation of (b);

[0022] (d) analyzing the results of the at least one perturbation toidentify genetic differences between the at least two genome-registeredcollections.

[0023] Additionally the invention provides a method for generating agenome-registered collection of reporter gene fusions comprising thesteps of:

[0024] (a) generating a set of gene fusions comprising:

[0025] 1) a reporter gene or reporter gene complex operably linked to

[0026] 2) a genomic fragment from an organism of which at least 15% ofthe genomic nucleotide sequence is known;

[0027] (b) introducing in vitro the reporter gene fusions from step (a)into a host organism;

[0028] (c) registering the reporter gene fusions on the basis ofsequence homology to the genomic sequence of the organism;

[0029] (d) repeating (a), (b), and/or (c) until reporter gene fusionshave been made to at least 15% of the known genomic nucleotide sequenceof said organism.

[0030] Similarly the invention provides a method for generating agenome-registered collection of reporter gene fusions comprising:

[0031] (a) generating random nucleic acid fragments from the DNA of anorganism of which at least 15% of the nucleotide sequence is known;

[0032] (b) operably linking the random nucleic acid fragments generatedin (a) to a vector containing a promoterless reporter gene or reportergene complex;

[0033] (c) introducing the vector (b) containing the gene fusions into ahost organism;

[0034] (d) determining the nucleic acid sequence of the distal and theproximal ends of the random nucleic fragments relative to the reportergene or reporter gene complex;

[0035] (e) registering the sequenced fusions of step (d) on the basis ofsequence homology to the genomic sequence of the host organism;

[0036] (d) repeating (a), (b), and/or (c) until reporter gene fusionshave been made to at least 15% of the known genomic nucleotide sequenceof said organism. Generation of the random nucleic acid fragments ofstep may incorporate restriction enzyme digestion, physical shearing ofthe genome and polymerase chain reaction.

[0037] In another embodiment the invention provides a method forgenerating a genome-registered collection of reporter gene fusionscomprising steps of:

[0038] (a) introducing one or more transposons into the genome of anorganism of which at least 15% of the nucleotide sequence is known, eachtransposon containing a promoterless reporter gene or reporter genecomplex;

[0039] (b) determining the nucleic acid sequence of the junction betweenthe proximal end of the genomic DNA and the transposon containing thereporter gene or reporter gene complex and registering the reporter genefusions relative to the genomic sequence of the organism,

[0040] (c) repeating (a) and (b) until reporter gene fusions have beenmade to at least 15% of the known genomic nucleotide sequence of saidorganism.

[0041] Alternatively the invention provides a method for identifying aprofile of inducing conditions for a reporter gene fusion comprising:

[0042] (a) obtaining a gene expression profile of an organism underinduced and non-induced conditions wherein induced genes are identified;

[0043] (b) providing a genome-registered collection of reporter genefusions, said fusions registered to the genome of the organism of (a);

[0044] (c) selecting the reporter gene fusions of (b) that correspond tothe induced genes of (a) to create a subset of the genome-registercollection;

[0045] (d) contacting the subset of the genome-register collection of(c) with the inducing conditions of (a) to identify at least onerepresentative reporter gene fusion whose expression was altered in asimilar manner as in (a);

[0046] (e) contacting the at least one representative reporter genefusion of (d) in a high throughput manner with a multiplicity ofdifferent inducing conditions to identify a profile of inducingconditions for that reporter gene fusion.

[0047] In another embodiment the invention provides a method forgenerating a genome-registered collection of reporter gene fusionscomprising:

[0048] (a) providing a genome from an organism wherein at least 15% ofthe nucleotide sequence is known;

[0049] (b) providing a series of amplification primers having homologyto specific known regions of the genome of (a);

[0050] (c) amplifying portions of the genome of (a) with the primers of(b) to create a collection of nucleic acid amplification products;

[0051] (d) operably linking the amplification products of (c) to avector containing a promoterless reporter gene or reporter gene complex;

[0052] (e) introducing the reporter gene fusions into a said organism;

[0053] (f) repeating (a)-(e) until, until reporter gene fusions havebeen made to at least 15% of the known genomic nucleotide sequence ofsaid organism.

[0054] In another embodiment the invention provides a method foridentifying a profile of inducing conditions for a reporter gene fusioncomprising:

[0055] (a) obtaining a gene expression profile for each of mutant strainand a parental strain organism under induced and non-induced conditionswherein induced genes are identified;

[0056] (b) providing a genome-registered collection of reporter genefusions, said fusions registered to the genome of the organism of (a);

[0057] (c) selecting the reporter gene fusions of (b) that correspond tothe induced genes of (a) to create a subset of the genome-registercollection;

[0058] (d) contacting the subset of the genome-register collection of(c) with the inducing conditions of (a) to identify at least onerepresentative reporter gene fusion whose expression was altered in asimilar manner as in (a);

[0059] (e) contacting the at least one representative reporter genefusion of (d) in a high throughput manner with a multiplicity ofdifferent inducing conditions to identify a profile of inducingconditions for that reporter gene fusion.

[0060] Similarly it is an object of the invention to provide a method tovalidate results from comprehensive genome analysis comprising the stepsof:

[0061] (a) analyzing a genome-wide, gene expression assay of an organismtreated with a condition or chemical of interest to identify genes withaltered expression;

[0062] (b) selecting from a genome-registered collection of reportergene fusions those reporter gene fusions containing promoter regionsoperably linked to genes corresponding to the altered genes from (a) orgenes co-regulated with genes corresponding to the altered genes from(a);

[0063] (c) testing expression of the reporter gene fusions selected from(b) with the conditions or chemicals of interest used in (a); and

[0064] (d) comparing the gene expression results from (c) to the geneexpression result of (a).

[0065] The invention additionally provides a method to determine operonstructure comprising steps of:

[0066] (a) selecting a subset of reporter gene fusions from agenome-registered collection of reporter gene fusions that map to theregion of a possible operon;

[0067] (b) assaying the subset for the reporter gene function; and

[0068] (c) determining a putative operon structure based on thequantities of reporter gene function.

[0069] Alternatively the invention provides a method for constructing acellular array containing reporter gene fusions comprising:

[0070] (a) generating a set of gene fusions comprising:

[0071] 1) a reporter gene or reporter gene complex operably linked to

[0072] 2) a genomic fragment from an organism of which at least 15% ofthe genomic nucleotide sequence is known;

[0073] (b) selecting a non-redundant subset of reporter gene fusionsfrom the set of (a) representative of at least 15% of known or suspectedpromoter regions from a genome-registered collection of reporter genefusions, each containing a known or suspected promoter region operablylinked to a reporter gene or reporter gene complex; and

[0074] (c) fixing the non-redundant subset of reporter gene fusions of(b) in an array format.

[0075] In a preferred embodiment the invention provides a method formeasuring gene expression responses to perturbation comprising:

[0076] (a) constructing at least 2 identical cellular arrays, eachcellular array comprising a reporter gene fusion comprising:

[0077] 1) a reporter gene or reporter gene complex operably linked to

[0078] 2) a genomic fragment from an organism of which at least 15% ofthe genomic nucleotide sequence is known;

[0079] wherein at least one cellular array is a control array and atleast one cellular array is an experimental array;

[0080] (b) contacting the experimental array of (a) with a perturbingcondition;

[0081] (c) comparing the differences between the gene expressionactivity of the control and the experimental array wherein geneexpression response to a perturbing condition is determined.

[0082] Organisms amenable to the present method include prokaryotes andfungi and particularly enteric bacterium.

[0083] Reporters useful in the present method include luxCDABE, lacZ,gfp, cat, galK, inaZ, luc, luxAB, bgaB, nptII, phoA, uidA and xylE.

BRIEF DESCRIPTION OF THE DRAWINGS

[0084]FIG. 1 is a diagram of fusions to the lac operon from the Lux-ACollection. The direction of the arrowhead indicates if the DNA of thecloned chromosomal segment is oriented such that a promoter, if present,would be driving expression of genes on the direct (arrowhead to theright) or complementary (arrowhead to the left ) strand.

[0085]FIG. 2 describes kinetics of induction of increasedbioluminescence from E. coli strain DPD3232 containing a yebF-luxCDABEfusion treated with various concentrations of MMC.

[0086] FIGS. 3(A-C) describes generally the Luxarray 0.5 luminescenceduring solid phase growth. FIG. 3A describes the cell placement toreplicate 96 well spacing to fit a microplate luminometer. In the whitelight on the left, each replicate of the twelve spots is outlined. Thebioluminescent image of the same plate is shown on the right. FIG. 3Bdescribes the signal collected for each clone as a function of time.Each cycle was about 20 min. FIG. 3C describes signals collected from 8replicates of same clone.

[0087] FIGS. 4(A-D) describes generally the Luxarray 0.5 perturbationwith nalidixic acid (NA). FIG. 4A represents results from strainscontaining the parental plasmid and two non-responding reporters, osmY,and lacZYA and FIG. 4B represents the results from three DNA damageresponsive reporters, uvrA, recA, dinG.

[0088]FIG. 5 describes High density Luxarray 0.5 perturbation withnalidixic acid. The squares represent reporter gene response fromnalidixic acid treated cultures and the circles represent reporter geneactivity from untreated cultures.

[0089]FIG. 6 describes graphical representation of selection criteriaimplemented to select Luxarray 1.0 Clones.

[0090]FIG. 7 describes bioluminescence captured from Luxarray 1.0reporters after 14 hr growth on LB media by cooled CCD array camera(FluorChem 8000, AlphaInnotech).

[0091]FIG. 8 describes the predicted promoters in a gene cluster of 20genes for typeI extracellular polysaccharide in E. coli and the promoteractivity of luxCDABE gene fusions from the region encoding production oftype I extracellular polysaccharide. The E. coli genes in this regionare shown with the top set of arrows. Above this line are the predictedpromoters regions from two sources (P, Blattner et al., (1997) Science277:1453-1462); P, Thieffry et al. (1998) Bioinformatics 14:391-400).The two promoters supported by DNA array and gene fusion data presentedhere are shown in bold type. The b designation and common name for eachgene is shown. The ratios of the deduced mRNA level in the rpoC mutantstrain to the deduced mRNA level in the rpoC⁺ strain determined by themicro array method are shown on the next line. The mapped location ofthe chromosomal inserts of selected luxCDABE gene fusions in this regionare shown with the second set of thicker arrows. Below these are the luxclone identification number and bioluminescence data (RLU/10⁹ cfu) foreach plasmid in the rpoC⁺ E. coli host strain and the host straincarrying an rpoC mutation. In the case of overlapping gene fusions, theshorter one is listed first.

[0092] FIGS. 9(A-D) represents generally the induction of the promoterb1728 by nalidixic acid (NA) in lexA+ host (FIG. 9A) in comparison tothe promoter in lexind host (FIG. 9C), and induction of the promoterb1728 by mitomicyn C (MC) in lexA+ host (FIG. 9B) in comparison to thepromoter in lexind host (FIG. 9D).

[0093]FIG. 10 represents uhpT-lux upregulation by limonene.

[0094] The invention can be more fully understood from the followingdetailed description and the accompanying sequence descriptions whichform a part of this application.

DETAILED DESCRIPTION OF THE INVENTION

[0095] The present invention relates to the generation and use of acellular array or a cellular array in combination with othergenome-registered arrays (an array of arrays) for the determination ofgene function and/or perturbation mode of action. Each cellular arrayconsists of a number of microbial strains. Each strain comprises onereporter gene fusion made up of a gene or gene fragment operably linkedto a reporter gene. Each gene or gene fragment has been “registered” ormapped to a specific location in the genome of the organism. Cellulararrays of the present invention are those that contain reporter genefusions to at least 15% of the genome of the organism being analyzed.Cellular arrays containing these reporter gene fusions are referred toherein as “registered collections”

[0096] The genome-registered collection of the invention may be used todetermine alterations in gene expression under a variety of conditions.Such collections are amenable to rapid assay and may be used to confirm,correct or augment data generated from DNA micro array technology.

[0097] In this disclosure, a number of terms and abbreviations are used.The following definitions are provided.

[0098] “Open reading frame” is abbreviated ORF. The term “ORF” is refersto a gene that specifies a protein.

[0099] “Polymerase chain reaction” is abbreviated PCR.

[0100] As used herein, an “isolated nucleic acid fragment” is a polymerof RNA or DNA that is single- or double-stranded, optionally containingsynthetic, non-natural or altered nucleotide bases. An isolated nucleicacid fragment in the form of a polymer of DNA may be comprised of one ormore segments of cDNA, genomic DNA or synthetic DNA.

[0101] The term “primer” refers to an oligonucleotide (synthetic oroccurring naturally), which is capable of acting as a point ofinitiation of nucleic acid synthesis or replication along acomplementary strand when placed under conditions in which synthesis ofa complementary stand is catalyzed by a polymerase. Wherein the primercontains a sequence complementary to a region in one strand of a targetnucleic acid sequence and primes the synthesis of a complementarystrand, and a second primer contains a sequence complementary to aregion in a second strand of the target nucleic acid and primes thesynthesis of complementary strand; wherein each primer is selected tohybridize to its complementary sequence, 5′ to any detection probe thatwill anneal to the same strand.

[0102] A “cellular array” means a set of strains differing from oneanother only in the chromosomal fragment fused to a reporter gene orreporter gene complex.

[0103] The term “array of arrays” refers to a collection of cellulararrays wherein each individual cellular array contains reporter genefusions responsive to a specific inducing condition or set ofconditions.

[0104] The terms “global scale” or “genome-wide scale” when applied toreporter gene fusions refer to a minimum 15% representation of thetranscription units of an organism. For example, there is predicted tobe 2328 transcription units (t.u.s) in E. Coli (RegulonDB Databasev.3.0,http://www.cifn.unam.mx/Computational_Biology/E.coli-predictions/). Thusa set of unique fusions representing 329 t.u.s (15% of 2328) is at“global scale”. The term “global scale” or “genome-wide scale” whenapplied to mutations or overexpression refers to a minimum 15%representation of the open reading frames of an organism.

[0105] The term “genome register” refers to a procedure of preciselylocating or mapping a defined nucleic acid sequence within a genome.

[0106] The term “genome-registered collection” refers to a set ofstrains containing reporter gene fusions, loss of gene functionmutations, altered gene function mutations, or overexpressed genes thathave been “registered” or mapped by homology to the nucleic acidsequence of the genome of the organism. These genome-registeredcollections include reporter gene fusions to at least 15%, morepreferably at least 20%, and most preferably at least 50% of all knownor predicted promoter regions, loss of gene function mutations in atleast 15%, more preferably at least 20%, and most preferably at least50%, of all known or predicted ORFs, altered gene function mutations inat least 15%, more preferably at least 20%, and most preferably at least50%, of all known or predicted ORFs, or overexpression of in at least15%, more preferably at least 20%, and most preferably at least 50%, ofall known or predicted ORFs.

[0107] As used herein the term “known” as applied to a gene or ORFwithin the context of a genome means that the sequence of the gene orORF is known and should not be limited to knowledge of the function ofthat gene or ORF.

[0108] The term “reporter gene fusion” refers to a chimeric geneconsisting, in one part, of a gene or genes that are useful to detecttranscription and/or translation initiated in the other part.

[0109] The term “reporter gene complex” refers to any set of two or moregenes that together are useful to generate a measurable signal.

[0110] The term “reporter gene activity” or “reporter constructactivity” refers to the accumulation of the limiting component(s) of thereporter system in use which results in the measurable signal associatedwith that reporter system. The measured activity is the sum result ofthe de novo production, ongoing degradation or deactivation, and/oractivation of the limiting component(s).

[0111] The term “Lux-A Collection” refers to a specific set of E. colistrains containing plasmid-borne luxCDABE fusions.

[0112] The term “lux fusion” or “luxCDABE fusion” refers to a chimericgene consisting, of a genomic sequence joined to luxCDABE genes.

[0113] The term “congenic strains” refer to two or more strains thatdiffer from one another by a single mutation or a strain differing inonly one gene to denote genes whose expression differ as a function ofthe allele.

[0114] The term “DNA microarray” or “DNA chip” means assembling PCRproducts of a group of genes or all genes within a genome on a solidsurface in a high density format or array. General methods for arrayconstruction and use are available (see Schena M, Shalon D, Davis R W,Brown P O., Quantitative monitoring of gene expression patterns with acomplementary DNA microarray. Science. Oct. 20, 1995; 270(5235): 467-70.and http://cmgm.stanford.edu/pbrown/mguide/index.html). A DNA microarrayallows the analysis of gene expression patterns or profile of many genesto be performed simultaneously by hybridizing the DNA microarraycomprising these genes or PCR products of these genes with cDNA probesprepared from the sample to be analyzed. DNA microarray or “chip”technology permits examination of gene expression on a genomic scale,allowing transcription levels of many genes to be measuredsimultaneously. Briefly, DNA microarray or chip technology comprisesarraying microscopic amounts of DNA complementary to genes of interestor open reading frames on a solid surface at defined positions. Thissolid surface is generally a glass slide, or a membrane (such as nylonmembrane). The DNA sequences may be arrayed by spotting or byphotolithography (see http://www.affymetrix.com/). Two separatefluorescently-labeled probe mixes prepared from the two sample(s) to becompared are hybridized to the microarray and the presence and amount ofthe bound probes are detected by fluorescence following laser excitationusing a scanning confocal microscope and quantitated using a laserscanner and appropriate array analysis software packages. Cy3 (green)and Cy5 (red) fluorescent labels are routinely used in the art, however,other similar fluorescent labels may also be employed. To obtain andquantitate a gene expression profile or pattern between the two comparedsamples, the ratio between the signals in the two channels (red:green)is calculated with the relative intensity of Cy5/Cy3 probes taken as areliable measure of the relative abundance of specific mRNAs in eachsample. Materials for the construction of DNA microarrays arecommercially available (Affymetrix (Santa Clara Calif.) Sigma ChemicalCompany (St. Louis, Mo.) Genosys (The Woodlands, Tex.) Clontech (PaloAlto Calif.) and Corning (Corning N.Y.). In addition, custom DNAmicroarrays can be prepared by commercial vendors such as Affymetrix,Clontech, and Corning.

[0115] The basis of gene expression profiling via micro-array technologyrelies on comparing an organism under a variety of conditions thatresult in alteration of the genes expressed. A single population ofcells may be exposed to a variety of stresses that will result in thealteration of gene expression. Alternatively, the cellular environmentmay be kept constant and the genotype may be altered. Typical stressesthat result in an alteration in gene expression profile will include,but is not limited to conditions altering the growth of a cell orstrain, exposure to mutagens , antibiotics, UV light, gamma-rays,x-rays, phage, macrophages, organic chemicals, inorganic chemicals,environmental pollutants, heavy metals, changes in temperature, changesin pH, conditions producing oxidative damage, DNA damage, anaerobiosis,depletion or addition of nutrients, addition of a growth inhibitor, anddesiccation. Non-stressed cells are used for generation of “control”arrays and stressed cells are used to generate an “experimental”,“stressed” or “induced” arrays. Induced arrays are those thatdemonstrate “altered gene expression”.

[0116] “Altered gene expression” refers to the change in the level of atranscription or translation products. If the gene is “up-regulated”,the level of transcription or translation products is elevated. If thegene is “down-regulated” the level of transcription or translationproducts is decreased. Conditions under which gene expression is alteredis also known as an “inducing condition”. In some instance a number ofdifferent conditions may result in the same transcriptional effect on asingle gene. Thus a number of inducing conditions will eitherup-regulate or down regulate the same gene. This collection of likeinducing conditions is known as a “profile of inducing conditions”.Similarly the term “perturbation” as used herein in reference to acellular array or a DNA micro array is any alteration of environment orgenotype the results in altered gene expression.

[0117] The terms “high density” or “comprehensive” micro array refers toa high-density DNA micro-array containing at least 75% of the openreading frames of the organism.

[0118] The term “expression profile” refers to the expression of groupsof genes.

[0119] The term “gene expression profile” refers to the expression ofindividual gene and suite of individual genes.

[0120] The “comprehensive expression profile” refers to the geneexpression profile of more than 75% of genes in the genome. In“comprehensive gene expression analysis” at least >75% of geneexpression of the organism is analyzed.

[0121] The term “corresponding to” is used herein to refer to similar orhomologous sequences, whether the exact position is identical ordifferent from the molecule to which the similarity or homology ismeasured. A nucleic acid or amino acid sequence alignment may includespaces. Thus, the term “corresponding to” refers to the sequencesimilarity, and not the numbering of the amino acid residues ornucleotide bases.

[0122] The terms “print” or “printing” refer to transferring one or morecultures from one locale, most often a multiwell culture plate, to asubstrate or solid surface by physical contact transfer or any of aplurality of technologies.

[0123] The term “regulon” refers to groups of operons sharing similarregulation.

[0124] The term “operon” refers to a unit of bacterial gene expressionand regulation, including structural genes and control elements in DNArecognized by regulator gene product(s) that in combination support theproduction of an mRNA.

[0125] The term “probe” refers to a single-stranded nucleic acidmolecule that can base pair with a complementary single stranded targetnucleic acid to form a double-stranded molecule.

[0126] The term “genotype” refers to the genetic constitution of anorganism as distinguished from its physical appearance.

[0127] The term “genomic DNA” refers to a single complete set of geneticinformation carried in the chromosomes of an organism.

[0128] The term “total RNA” refers to non-fractionated RNA from anorganism.

[0129] The terms “protein specifying RNA” or “protein specifyingtranscript” refer to RNA derived from an ORF.

[0130] The terms “transposon” and “transposable element” are usedinterchangeably and mean a region of nucleic acid that is capable ofmoving from one position to another where this movement is catalyzed bythe element itself.

[0131] The term “transposition” means a biochemical reaction thatcatalyzes the movement of a transposable element from one site intodifferent site within a DNA molecule. Transposition can be carried outin vivo or in vitro.

[0132] The term “in vitro transposition” means a biochemical reactioninitiated outside the cell that catalyzes the movement of a transposableelement from one site into different site within a DNA molecule.

[0133] The term “in vivo transposition” means a biochemical reactionthat takes place within the cell that catalyzes the mobilization of atransposon from of site to another within the genome of the host.

[0134] A nucleic acid molecule is “hybridizable” to another nucleic acidmolecule, such as a cDNA, genomic DNA, or RNA, when a single strandedform of the nucleic acid molecule can anneal to the other nucleic acidmolecule under the appropriate conditions of temperature and solutionionic strength. Hybridization and washing conditions are well known andexemplified in Sambrook, J., Fritsch, E. F. and Maniatis, T. MolecularCloning: A Laboratory Manual, Second Edition, Cold Spring HarborLaboratory Press, Cold Spring Harbor (1989), particularly Chapter 11 andTable 12.1 therein (entirely incorporated herein by reference). Theconditions of temperature and ionic strength determine the “stringency”of the hybridization. Hybridization requires that the two nucleic acidscontain complementary sequences, although depending on the stringency ofthe hybridization, mismatches between bases are possible. Theappropriate stringency for hybridizing nucleic acids depends on thelength of the nucleic acids and the degree of complementation, variableswell known in the art. The greater the degree of similarity or homologybetween two nucleotide sequences, the greater the value of Tm forhybrids of nucleic acids having those sequences. The relative stability(corresponding to higher Tm) of nucleic acid hybridizations decreases inthe following order: RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greaterthan 100 nucleotides in length, equations for calculating Tm have beenderived (see Sambrook et al., supra, 9.50-9.51). For hybridizations withshorter nucleic acids, i.e., oligonucleotides, the position ofmismatches becomes more important, and the length of the oligonucleotidedetermines its specificity (see Sambrook et al., supra, 11.7-11.8).Furthermore, the skilled artisan will recognize that the temperature andwash solution salt concentration may be adjusted as necessary accordingto factors such as length of the probe.

[0135] The term “complementary” is used to describe the relationshipbetween nucleotide bases that are capable to hybridizing to one another.For example, with respect to DNA, adenosine is complementary to thymineand cytosine is complementary to guanine. Accordingly, the instantinvention also includes isolated nucleic acid fragments that arecomplementary to the complete sequences as reported in the accompanyingSequence Listing as well as those substantially similar nucleic acidsequences.

[0136] “Gene” refers to a nucleic acid fragment that expresses aspecific protein, including regulatory sequences preceding (5′non-coding sequences) and following (3′ non-coding sequences) the codingsequence, unless mentioned otherwise. For example, “reporter genes” usedin gene fusion does not include regulatory (promoter) sequences unlessspecified otherwise. “Native gene” refers to a gene as found in naturewith its own regulatory sequences.

[0137] The terms “chimeric gene”, “gene fusion”, or “fusion” refer toany non-native gene or genes comprising two or more genomic orartificial DNA fragments that are not found in nature. Accordingly, achimeric gene, gene fusion or fusion may comprise regulatory sequencesand coding sequences the are derived from different sources, orregulatory sequences and coding sequences derived from the same sourcebut arranged in a manner that different than that is found in nature.“Endogenous gene” refers to a native gene in its natural location in thegenome of an organism. A “foreign” gene refers to a gene not normallyfound in the host organism, but that is introduced into the hostorganism by gene transfer. Foreign genes can comprise native genesinserted into a non-native organism, or chimeric genes. A “transgene” isa gene that has been introduced into the genome by a transformationprocedure.

[0138] The term “coding sequence” refers to a DNA sequence that codesfor a specific amino acid sequence. “Suitable regulatory sequences”refer to nucleotide sequences located upstream (5′ non-codingsequences), within, or downstream (3′ non-coding sequences) of a codingsequence, and which influence the transcription, RNA processing orstability, or translation of the associated coding sequence. Regulatorysequences may include promoters, translation leader sequences, introns,and polyadenylation recognition sequences.

[0139] The term “promoter” refers to a DNA sequence to which RNApolymerase can bind to initiate the transcription. In general, a codingsequence is located 3′ to a promoter sequence. Promoters may be derivedin their entirety from a native gene, or be composed of differentelements derived from different promoters found in nature, or evencomprise synthetic DNA segments. It is understood by those skilled inthe art that different promoters may direct the expression of a gene indifferent tissues or cell types, or at different stages of development,or in response to different environmental conditions. Promoters whichcause a gene to be expressed in most cell types at most times arecommonly referred to as “constitutive promoters”. It is furtherrecognized that since in most cases the exact boundaries of regulatorysequences have not been completely defined, DNA fragments of differentlengths may have identical promoter activity. “Promoter region” ispromoter and adjacent areas whose function may be modulate promoteractivity.

[0140] The “3′ non-coding sequences” refer to DNA sequences locateddownstream of a coding sequence and include polyadenylation recognitionsequences and other sequences encoding regulatory signals capable ofaffecting mRNA processing or gene expression. The polyadenylation signalis usually characterized by affecting the addition of polyadenylic acidtracts to the 3′ end of the mRNA precursor.

[0141] “RNA transcript” refers to the product resulting from RNApolymerase-catalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect complementary copy of the DNA sequence, it isreferred to as the primary transcript or it may be a RNA sequencederived from post-transcriptional processing of the primary transcriptand is referred to as the mature RNA. “Messenger RNA (mRNA)” refers tothe RNA that is without introns and that can be translated into proteinby the cell. “cDNA” refers to a double-stranded DNA that iscomplementary to and derived from mRNA. “Sense” RNA refers to RNAtranscript that includes the mRNA and so can be translated into proteinby the cell. “Antisense RNA” refers to a RNA transcript that iscomplementary to all or part of a target primary transcript or mRNA andthat blocks the expression of a target gene (U.S. Pat. No. 5,107,065).The complementarity of an antisense RNA may be with any part of thespecific gene transcript, i.e., at the 5′ non-coding sequence, 3′non-coding sequence, introns, or the coding sequence. “Functional RNA”refers to antisense RNA, ribozyme RNA, or other RNA that is nottranslated yet has an effect on cellular processes.

[0142] The term “operably linked” refers to the association of nucleicacid sequences on a single nucleic acid fragment so that the function ofone is affected by the other. For example, a promoter is operably linkedwith a coding sequence when it is capable of affecting the expression ofthat coding sequence (i.e., that the coding sequence is under thetranscriptional control of the promoter). Coding sequences can beoperably linked to regulatory sequences in sense or antisenseorientation.

[0143] The term “expression”, as used herein, refers to thetranscription and stable accumulation of sense (mRNA) or antisense RNAderived from the nucleic acid fragment of the invention. Expression mayalso refer to translation of mRNA into a polypeptide.

[0144] The term “transformation” refers to the acquisition of new genesin a cell after the incorporation of nucleic acid.

[0145] The terms “plasmid”, “vector”, and “cassette” refer to an extrachromosomal element often carrying genes which are not part of thecentral metabolism of the cell, and usually in the form of circulardouble-stranded DNA molecules. Such elements may be autonomouslyreplicating sequences, genome integrating sequences, phage or nucleotidesequences, linear or circular, of a single- or double-stranded DNA orRNA, derived from any source, in which a number of nucleotide sequenceshave been joined or recombined into a unique construction which iscapable of introducing a promoter fragment and DNA sequence for aselected gene product along with appropriate 3′ untranslated sequenceinto a cell. “Transformation cassette” refers to a specific vectorcontaining a foreign gene and having elements in addition to the foreigngene that facilitate transformation of a particular host cell.“Expression cassette” refers to a specific vector containing a foreigngene and having elements in addition to the foreign gene that allow forenhanced expression of that gene in a foreign host.

[0146] The term “restriction endonuclease” or “restriction enzyme”refers to an enzyme which binds and cuts within a specific nucleotidesequence within double stranded DNA.

[0147] The term “bioluminescence” refers to the phenomenon of lightemission from any living organism.

[0148] The term “relative light unit” is abbreviated “RLU” and refers toa measure of light emission as measured by a luminometer, calibratedagainst an internal standard unique to the luminometer being used.

[0149] The term “stress” or “environmental stress” refers to thecondition produced in a cell as the result of exposure to anenvironmental insult.

[0150] The terms “insult” or “environmental insult” refers to anysubstance or environmental change that results in an alteration ofnormal cellular metabolism in a bacterial cell or population of cells.Environmental insults may include, but are not limited to, chemicals,environmental pollutants, heavy metals, changes in temperature, changesin pH as well as agents producing oxidative damage, DNA damage,anaerobiosis, changes in nitrate availability or pathogenesis.

[0151] The term “stress response” refers to the cellular responseresulting in the induction of either detectable levels of stressproteins or in a state more tolerant to exposure to another insult or anincreased dose of the environmental insult.

[0152] The term “stress gene” refers to any gene whose transcription isinduced as a result of environmental stress or by the presence of anenvironmental insults.

[0153] The terms “log phase”, “log phase growth”, “exponential phase”,or “exponential phase growth” refer to cell cultures of organismsgrowing under conditions permitting the exponential multiplication ofthe cell number.

[0154] Standard recombinant DNA and molecular cloning techniques usedhere are well known in the art and are described by Sambrook, J.,Fritsch, E. F. and Maniatis, T., Molecular Cloning: A Laboratory Manual,Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y. (1989); and by Silhavy, T. J., Bennan, M. L. and Enquist, L. W.,Experiments with Gene Fusions, Cold Spring Harbor Laboratory Cold PressSpring Harbor, N.Y. (1984); and by Ausubel, F. M. et al., CurrentProtocols in Molecular Biology, published by Greene Publishing Assoc.and Wiley-Interscience (1987).

[0155] In this invention, a random set of fragments of E. coli genomewas generated by partially digesting E. coli genome with the restrictionenzyme Sau3A1, and separating the fragments by size fractionation. Thefraction with an average size of 1.8 KB was isolated and ligated to theplasmid pDEW201 which contains the origin of replication and bla frompBR322, four transcription terminators upstream of the promoterlessluxCDABE gene. The ligation products were transformed into E. coliXL2Blue cells. Approximately 24000 resulting transformants were pooledand used AS the source of heterologous plasmid DNA. This plasmid DNApool was used to transform E. coli DPD1675 cells. A total of 8066individual transformants were isolated and labeled as Lux-A Collection.A homology search for the sequence from beginning and end of clones fromLux-A Collection was performed against complete E. coli sequence. Thelocation and orientation of each chimeric gene fusion with respect tothe E. coli genome was determined based on the computed homology.Additional sequences for the collection can be added by more randomfusions or inverting the orientation of DNA in the fusion to put thepromoter regions in the right orientation for the inserted DNA by usingthe method that is routine in the art.

[0156] Preferred organisms for use in the present invention are thosewhose genomes are being sequenced or that have completely sequencedgenomes and are amenable to introduction of gene fusions bytransduction, transformation, or conjugation. They include but are notlimited to the 113 organisms listed on the WEB page,http://www.ncbi.nlm.nih.gov/PMGifs/Genomes/bact.html as of Feb. 15,2000. In addition to the organisms mentioned above, any microbialorganism with at least 15%, preferably 20%, and most preferably 50% ofits genome sequence known that is amenable to introduction of genefusions by transduction, transformation, or conjugation can be used inthis invention to generate reporter gene fusions.

[0157] Within the context of the present invention prokaryotes and fungihaving at least 15% of their genome sequence are particularly suitable.Of the prokaryotes, enteric bacteria such as Escherichia and Salmonellaare preferred and E. coli is most preferred as it is well characterizedand its genome has been sequenced. it is most preferred prokaryoticorganism for this invention.

[0158] It will be appreciated that for the purposes of the presentinvention that the teachings with respect to E. coli are particularlyadaptable to any of the enteric bacteria. Enteric bacteria are membersof the family Enterobacteriaceae, and include such members asEscherichia, Salmonella, and Shigella. They are gram-negative straightrods, 0.3-1.0×1.0-6.0 μm, motile by peritrichous flagella, except forTatumella, or nonmotile. They grow in the presence and absence of oxygenand grow well on peptone, meat extract, and (usually) MacConkey's media.Some grow on D-glucose as the sole source of carbon, whereas othersrequire vitamins and/or mineral(s). They are chemoorganotrophic withrespiratory and fermentative metabolism but are not halophilic. Acid andoften visible gas is produced during fermentation of D-glucose, othercarbohydrates, and polyhydroxyl alcohols. They are oxidase negative and,with the exception of Shigella dysenteriae 0 group 1 and Xenorhabdusnematophilus, catalase positive. Nitrate is reduced to nitrite except bysome strains of Erwinia and Yersina. The G+C content of DNA is 38-60 mol% (T_(m), Bd). DNAs from species from species within most genera are atleast 20% related to one another and to Escherichia coli, the typespecies of the family. Notable exceptions are species of Yersina,Proteus, Providenica, Hafnia and Edwardsiella, whose DNAs are 10-20%related to those of species from other genera. Except for Erwiniachrysanthemi all species tested contain the enterobacterial commonantigen (Bergy's Manual of Systematic Bacteriology, D. H. Bergy, et al.,Baltimore: Williams and Wilkins, 1984).

[0159] As for the reporter gene or gene complex, luxCDABE is mostpreferred for its sensitivity and simplicity of assay conditions. Otherreporter genes well known in the art also can be used in this invention.The preferred reporter genes include but are not limited to lacZ, gfp,cat, galK, inaZ, luc, luxAB, bgaB, nptII, phoA, uidA, and xylE.

[0160] Methods to introduce fusions into such strains are well known inthe art. Fusions can be introduced by transduction, transformation orconjugation as appropriate for each specific organism. They can beconstructed in vitro by techniques including but not limited totransposon (e.g., Tn7) mediated transposition, ligating of randomfragments to a reporter gene construct containing a vector, or ligatingof PCR products to the same vector. They can be constructed by in vivotransposition where the transposon is introduced into the cell bytransduction, transformation, or conjugation. Kits for in vitrotransposition are commercially available (see for example The PrimerIsland Transposition Kit, available from Perkin Elmer AppliedBiosystems, Branchburg, N.J., based upon the yeast Ty1 element(including the AT2 transposon); The Genome Priming System, availablefrom New England Biolabs, Beverly, Mass.; based upon the bacterialtransposon Tn7; and the EZ::TN Transposon Insertion Systems, availablefrom Epicentre Technologies, Madison, Wis., based upon the Tn5 bacterialtransposable element.

[0161] The preferred methods to generate a set of fragments of thegenome include but are not limited to, the partial digestion with arestriction enzyme, physical shearing, polymerase chain reaction (PCR)amplification, the combination of restriction enzyme digestion and PCR,the combination of restriction enzyme digestion and physical shearing,the combination of physical shearing and PCR, and the combination of allthree. Above methods for generating fragments are well known in the art.For example PCR is well described in (U.S. Pat. No. 4,683,202 (1987,Mullis, et al.) and U.S. Pat. No. 4,683,195 (1986, Mullis, et al.), andrestriction enzyme digestion and physical shearing is well described inSambrook et al., supra.

[0162] When using PCR to generate fragments several approaches may betaken. In one instance random primers may be used to amplify portions ofthe genome. In this situation the fragments, (amplification products)generated will be random in nature. In the alternative, where portionsof the sequence of the genome are known primers may be designed tospecific loci within the genome. This is a directed fragment generationapproach.

[0163] Furthermore, chimeric reporter gene fusions can be also made byusing transposition at the genome level. The above-described methods forgenerating transpositions are well known in the art.

[0164] After the sequencing and genome-registering the majority of theLux-A Collection members were completed the biological responses of thestrain collection containing luxCDABE fusions to members ofwell-characterized regulatory circuits were examined.

[0165] Thus, gene fusions to the lac operon, and members of the heatshock, SOS, SoxRS, and OxyR regulons were selected from the Lux-ACollection and the responses to known inducers of each of these globalregulatory circuits were determined.

[0166] Appropriate biological responses were demonstrated for each ofregulons mentioned above. The Lux-A Collection contained 3 members thatare fusions of luxCDABE to the lac operon (FIG. 1). These cultures weregrown in the presence of glucose and bioluminescence was measured andcompared to the cultures grown in the absence of glucose. Cultures grownin the absence of glucose were much more highly bioluminescent than theones in the presence of glucose.

[0167] Four out of twelve heat shock promoters were found as luxfusions. A total of 6 fusion containing strains were isolated and testedfor the stress response in the presence of various concentration ofethanol. The heat shock regulon gene fusions from the Lux-A Collectionconfirmed induction of heat shock response by ethanol.

[0168] For SOS regulon, 7 lux fusion were found in the Lux-A Collection.When these stains were tested with DNA damaging chemical nalidixic acid,increased level of bioluminescence was observed. However, treatment withethanol did not induce bioluminescence in the same culture.

[0169] In a similar fashion, although SoxR/S and OxyR regulons were notfully represented in the Lux-A Collection, the biologically appropriateresponse was observed.

[0170] Thus, while the Lux-A Collection is not comprehensive incontaining a fusion controlled by each promoter in E. coli, itnonetheless provides a genomic-wide overview of transcriptionalresponses to imposed stresses and can be adapted to optimize responses.

[0171] The effect of mitomycin C (MMC), a known DNA damaging agent, wastested with DNA microarray and the resulting gene expression pattern wascompared with the gene expression data from the treated cells of theLux-A Collection. As expected, the expression of the known SOS genes waselevated in micro array data. However the expression of several SOSregulon genes was elevated less than 2-fold (Table 12) and as such werewithin a large group of 792 genes the expression of which was elevatedby 20% or more. Because most of these are likely due to artifacts in thearray data rather than to actual biologically relevant responses, thegroup of genes with less than two fold increase in expression wereconsidered MMC non-inducible genes. Had these experiments been conductedusing a compound with an otherwise unknown mode of action, somebiologically relevant gene expression events would have been missed withthe DNA micro array approach. To test if strains carrying luxCDABE genefusions would yield the expected positive result, the three gene fusionsthat were available in the Lux-A Collection of reporter gene fusionswere tested for mitomycin C responses. In all three cases, mitomycin Cinduced increased bioluminescence. Thus, this demonstrates that negativeresults from DNA microarrays can be questioned by contradictory positiveresults with corresponding gene fusions.

[0172] The genes not previously known to be upregulated with mitomycinC, provide an opportunity to further examine the correlation of DNAarray and gene fusion experimental data. For this class of genes, fourfusions were available in the Lux-A Collection of reporter gene fusions.The corresponding luxCDABE fusions to these four genes provided noevidence of increased gene expression induced by MMC. Thus, the positiveresults from the array were classified as false-positive.

[0173] One gene fusion in the Lux-A Collection of gene fusions was foundto have a genomic fragment that when inverted would result in a fusionto yebF, a gene observed to be upregulated by mitomycin C in DNA microarray experiments. When the genomic DNA fragment was released andinserted back into the vector, bioluminescence from some of thetransformed colonies were found to be highly inducible by another DNAdamaging agent, nalidixic acid, strongly suggesting that the inversionof DNA might have occurred. Furthermore, induction of bioluminescence byMMC was demonstrated as shown in FIG. 2.

[0174] The induction of increased gene expression from the yebF-luxfusion not only validates the data from the DNA array study, butfurthermore, allows facile dose response and kinetic analyses andprovides a biosensor strain for a high throughput screen. It is possiblethat the DNA damage response reported by yebF-lux fusion might bemediated by the well-characterized SOS response because there is a LexAbox upstream of yebG (Lomba et al., (1997) FEMS Microbiol Lett156:119-122), which is just upstream of yebF. However, there is alsosuggested to be a promoter that drives transcription of the yebF gene(Blattner et al., (1997) Science 277:1453-1462), therefore the inductionof yebF expression by MMC was unexpected. Thus, these resultsdemonstrate the concept that a previously unknown gene expression eventcan be discovered with a DNA microarray, then a corresponding lux genefusion can be used to validate and extend the results. Furthermore, sucha lux fusion strain can form the basis of a high throughput screen basedon gene expression changes.

[0175] The yebF-luxCDABE gene fusion mentioned in the Example 3 can beused as a high throughput screen for compounds other than MMC andnalidixic acid that result in DNA damage. The use of the luxCDABEbioluminescent reporter fusion allows facile detection of reporter geneactivity in a manner that does not require cell lysis or addition ofenzymatic substrates. Thus development of a high throughput screen onlyrequires an instrument to quantitate light production (of which thereare many available commercially) and a source of bacterial cell culturescontaining the gene fusion of choice. Such bacterial cell cultures canbe supplied by one of several methods. A basic way to use bacterialstrains containing gene fusions is simply with freshly grown bacterialcell cultures. An alternative to daily cultivation is the use of frozenculture aliquots, such as has been successfully demonstrated with an E.coli bioluminescent sensor that is competent for gene expression assaysimmediately after thawing, having been previously stored at −80° C.using a cryoprotectant such as glycerol (Sticher et al., (1997) Appl.Environ. Microbiol. 63:4053-4060). Two other common methods of handlingbacteria, lyophilization and continuous culture, have also proven to beuseful sources of lux fusion strains for testing purposes.Lyophilization has been successfully applied to metal responsive andheat shock responsive cellular biosensor strains (Corbisier et al.,(1996) Environ. Toxicol. Water Qual. 11:171-177; Tauriainen et al.,(1998) Biosensors & Bioelectronics 13:931-938; Tauriainen et al., (1997)Appl. Environ. Microbiol. 63:4456-4461; Van Dyk and Wagner (1998) U.S.Pat. No. 5,731,163; Wagner and Van Dyk (1998), Methods in MolecularBiology: Bioluminescence Methods and Protocols, vol. 102. Humana PressInc p.: 123-127). Continuous cultivation of E. coli bioluminescentbiosensors in mini bioreactors has been shown to yield reproducibledetection of stress responses (Gu et al., (1996), Biotechnol. Prog.12:393-397; Gu et al., (1999) Biosens. Bioelectron. 14:355-361).

[0176] Other possible methods to supply cellular biosensors for highthroughput screens are immobilization by entrapment in a carriermaterial or use of a bioluminescent bioreporter integrated circuit(BBIC). Strontium alginate immobilization of a lux fusion containingbacterial strain has been demonstrated for use as a probe for wastestreams (Heitzer et al., (1994) Appl. Environ. Microbiol. 60:1487-1494;Matrubutham et al., (1997) Appl. Microbiol. Biotechnol. 47:604-609; Webbet al, (1997) Biotechnol. Bioeng. 54:491-502). In another example ofimmobilization, calcium alginate beads, harboring an SOS responsive luxfusion strain, stored in a CaCl₂ solution at 4° C. were found to giveuseful DNA damage induction responses for up to one month afterformation (Davidov et al., in press). Likewise, calcium alginateimmobilization of a copper-responsive biosensor results in superiorstability of the biosensor relative to the immobilization of the samebiosensor in agarose (de Lorenzo et al., (1999) Anal. Chim. Acta387:235-244). Furthermore, combining of immobilized cells and lightdetection equipment is possible for sensors that produce visible lightas a signal. In one such case, an E. coli luc fusion strain isimmobilized on the end of fiber optic monitoring device (Ikariyama etal., (1997) Anal. Chem. 69:2600-2605). Finally, in the BBIC approach,which takes advantage of the cellular signal generated by the five geneluxCDABE reporter, a bioluminescent biosensor strain is deposited onto amicro-luminometer fabricated within an integrated circuit; the lightproduced by the biosensor is detected by the integrated circuit, whichthen processes and communicates the results (Simpson et al., (1998) Soc.Opt. Eng. 3328 (Smart Electronics and MEMS):202-212; Simpson et al.,(1998) TIBTECH 16:332-338).

[0177] Therefore, development of a DNA damage responsive high throughputscreen based on the newly discovered yebF-luxCDABE fusion is readilyaccomplished by choosing one of the above known methods to provide anactive cellular biosensor and combining it, if necessary, with aninstrument that measures visible light production. Other promoters froma stress response gene may be used to generate high throughput screensusing a lux fusion. Stress response gene promoters from both prokaryoticand eukaryotic cells may be used, however promoters from bacteria arepreferred and promoters from E. coli are most preferred. Suitable stressresponse gene promoters may be selected from but are not limited to thelist of genes under the heading “responding genes” given in Table 1below, and other newly discovered regulatory circuits. TABLE 1REGULATORY REGULATORY RESPONDING STIMULUS GENE(S) CIRCUIT GENES* ProteinrpoH Heat Shock grpE, dnaK, Damage^(a) lon, rpoD, groESL, lysU, htpE,htpG, htpI, htpK, clpP, clpB, htpN, htpO, htpX, etc. DNA Damage^(b)lexA, recA SOS recA, uvrA, lexA, umuDC, uvrA, uvrB, uvrC, sulA, recN,uvrD, ruv, dinA, dinB, dinD, dinF etc. Oxidative oxyR Hydrogen katG,ahp, Damage^(c) Peroxide etc. Oxidative soxRS Superoxide micF, sodA,Damage^(d) nfo, zwf, soi, etc. Membrane fadR Fatty Acid fabA Damage^(e)Starvation Any^(f) ? Universal uspA Stress Stationary rpoS Resting StatexthA, katE, Phase^(g) appA, mcc, bolA, osmB, treA, otsAB, cyxAB, glgS,dps, csg, etc. Amino Acid relA, spoT Stringent his, ilvBN,Starvation^(h) ilvGMEDA, thrABC, etc. Carbon cya, crp Catabolite lac,mal, gal, Starvation^(i) Activation ara, tna, dsd, hut, etc. PhosphatephoB, phoM, P Utilization phoA, phoBR, Starvation^(j) phoR, phoU phoE,phoS, aphA, himA, pepN, ugpAB, psiD, psiE, psiF, psiK, psiG, psiI, pstJ,psiN, psiR, psiH, phiL, phiO, etc. Nitrogen glnB, glnD, N UtilizationglnA, hut, Starvation^(k) glnG, glnL etc.

[0178] Fusions to lux reporter genes can also be used to supportidentification of the operonic structures, newly discovered regulatorycircuits, and promoter sites. Since the random library of E. coligenomic DNA was fused to the promoterless lux gene, the strength ofbioluminescence in the resulting fusions should depend on the promoterfrom the E. coli. For example, if the fusion contains the completepromoter, lux gene expression would be stronger than the fusion thatcontains a partial region of same promoter. If the promoter is truncatedto the point that it is non-functional, the lux gene expression would beabsent. Such result can support the postulated operonic structures anddependence of promoter function in host (Example 4).

[0179] The construction and sequencing of a random library of E. coligenomic fragments in plasmid pDEW201 has generated a genome-registeredset of promoter activity reporter constructs. A highly parallel, solidphase cellular assay has been developed utilizing a subset of thesereporters by combining bioinformatic analysis and robotic manufacturingof arrays and high throughput culture production. This assay has thecapacity, utilizing standard robotic tools, to monitor promoter activityof entire microbial genomes in duplicate, or alternatively entiregenomes of different microbes on the same array. The output of the assayis a grayscale image and is compatible with commercial products forimage analysis and data analysis developed specifically of DNAmicroarray technologies.

[0180] Briefly, the assay involves creating a nonredundant collection ofclones containing reporter constructs. These cells are grown tostationary phase and robotically printed at high density onto a porousmembrane (e.g., Biodyne B Nunc). Any contact or non contact printingrobot (e.g., Biomek 2000, Beckman) may be used for printing. Themembrane is in close contact with solid media. A key aspect of thisinvention is the ability to move the membrane from one surface toanother surface containing different media. The ability to measure theluminescence as a function of regulatory region activity from the cellsgrown in the solid surface was surprising and unexpected. Previously,LaRossa and Van Dyk (U.S. Pat. No. 6,025,131) reported that the methodto detect the activity of regulatory regions as reporter gene activityin suitable hosts was restricted to the cells grown in the liquid media.Growth in solid media allows one to grow the cells to a desired densityprior to perturbation then follow the kinetics of the response.Experimental protocols often involve perturbations that prohibit longterm exposure due to cell death or other irreversible effects. Theability to move the entire array to new growth conditions allows one agreat variety of experimental schemes including, but not limited, topulsed or pulse/chase exposures, reversibility, and short term kineticstudies. Effects of perturbants can be determined by comparison ofluminescence generated by treated and control cultures.

[0181] Several important characteristics of the assay system needed tobe evaluated. In particular, growth density and conditions, sensitivity,reproducibility, and the ability to perturb the reporters and detectchanges were major focus points.

[0182] Using DNA damage in E. Coli as a model system, the assay wasfirst developed with a small set of well characterized clones toevaluate the robustness of the approach. The set of 10 clones weretested for the response to DNA damaging agent, nalidixic acid. Theexpected nalidixic induced upregulation of genes in the SOS regulon wasdetected as increased bioluminescence (FIG. 4B). The level ofbioluminescence from the strains containing fusions to genes that arenot responsive to DNA damage were not affected by the nalidixic acidtreatment (FIG. 4A).

[0183] The clone set of Luxarray 0.5 was used to develop a highlyparallel solid phase assay by printing the clones at high density. Thisinvention allows collection of an image of the signal generated fromreporter constructs such that the signal intensity can be subsequentlyquantified. This requires not only that the collection parameters (focalplane, magnification, integration time, and algorithm) are constant butalso that the downstream image analysis software has the ability toprocess the images generated. Chemical perturbation, one of the utilityof this invention, requires physically relocating a membrane from oneculture plate to another. This results in images with minimal X-Ypositional registration. Several commercially available products canefficiently process these images. ArrayVision™ (Imaging Research,Toronto, Canada) and ImageQuant (Molecular Dynamics, Sunnyvale, Calif.)are two examples of appropriate software packages. It is preferred thata cooled CCD camera is used to capture the data.

[0184] Computational filters were applied to the genome-registered clonecollection to generate a nonredundant set of reporter constructsrepresenting approximately 28% of the promoters in the genome. Even atthis level of coverage, it is likely that at least one representativepromoter of all the regulons in E. coli is present in the collection.This large set was used in DNA damage perturbation experiments toconfirm the response of several promoters. As found with the low densityexperiments quantified with the luminometer, the expected responses foreach clone were well demonstrated. The five documented DNAdamage-responsive reporter constructs clearly show an upregulation ofexpression. In contrast, light production from the strain carrying thelac promoter fusion as well as several strains carrying other promoterfusions was decreased.

[0185] A high density cellular array (Lux array 1.0) in this inventionwas also used to identify a number of putative previously unknown DNAdamage-responsive operons. When treated with the DNA damaging chemical,nalidixic acid, in addition to the expected promoter activity (i.e., SOSregulon), several previously undescribed promoters demonstrated asimilar behavior. This invention can be used to monitor transcriptionalchanges in high throughput manner.

[0186] This invention also provides a method to identify the fusionsthat would be useful except that the orientation of the chromosomal DNAis inverted relative to the reporter genes, such that the promoterregions of interest are not operably linked to the reporter genes.Selecting such fusions and inverting the orientation of the insert DNAcan significantly enhance the utility of a sequenced collection byadding many more operable linked fusion to the collections. A simple wayto do this is to digest the plasmid DNA with a single restriction enzymethat cuts just outside the cloned region and religate the pieces.Although a mixture of plasmids results from this procedure, in manycases the correctly oriented plasmid can be found because cellscontaining it, but not other possible products, will produce light. Thisinverting method was also used to add gene fusions to the Lux-ACollection. Other methods besides light production can be used (notablyPCR) to identify the orientation or size of the insert.

[0187] Additionally, specific PCR primers can be designed for any regionof interest on the chromosome for which sequence data is available. ForE. coli the entire genome is available. Therefore by utilizingpositional and directional coordinates for known and predictedpromoters, specific primers were designed for the purpose of generatinga PCR product containing each promoter. The actual promoter was assumedto lie in a 400 basepair genomic fragment ending at the translationalstart codon position of the first open reading frame in the operon.Primer pairs were selected from this region for each operon such thatthe “right” primer was forced to include the start codon of the firstopen reading frame of the operon (Primer3, Rozen and, Skaletsky(1996,1997,1998). Code available athttp://www-genome.wi.mit.edu/genome_software/other/primer3.html).Acceptable PCR primer pairs were identified for 80% of the operons usingthe default Primer3 parameters with an optimal melting temperature, Tm,of 68° C. Primer pairs for the remaining operons can be identified byeither relaxing one or more parameters, expanding the search area or byusing a different algorithm. The final version of the primers alsoincludes addition of appropriate EcoRI and SacI endonuclease cleavagesites (left and right primers respectively) such that the PCR productscan be directionally cloned into pDEW201.

[0188] Still other methods to generate gene fusions for agenome-registered collection result in chromosomal rather thanplasmid-borne gene fusions. Transposable genetic elements carryingreporter genes can be used to generate gene fusions. If this is done byrandom transposition into the chromosome of the host organism, thesubsequent gene fusions can be genome-registered with respect to thechromosomal DNA sequence by determining the sequence of the junctionbetween the transposable element and the hromosome (Nichols et al.,(1998) J. Bacteriol. 180:6408-6411). Alternatively, transpositions donein vitro using a defined (and thus genome-registered) segment ofchromosomal DNA can be recombined by homologous recombination into thehost chromosome. Furthermore, gene fusions formed in vitro by othermethods, such as clones in plasmid DNA, can be recombined into thechromosome of the host by homologous recombination (Balbas et al.,(1996) Gene 172:65-69; Lloyd and Low (1996) In Escherichia coli andSalmonella: Cellular and Molecular Biology. ASM Press, pp 2236-2255),site specific recombination (Nash H. (1996) In Escherichia coli andSalmonella: Cellular and Molecular Biology. ASM Press, pp 2363-2376) orboth (Boyd et al., (2000) J. Bacteriol. 182:842-847).

[0189] The preferred methods to generate gene fusions include are butnot limited to the use of plasmid DNA from clones requiringreorientation as template for the PCR to generate inversion, use ofspecific PCR primers when sequence data is available, or use oftransposition to generate gene fusions in chromosome (Kenyon and Walker(1980) Proc. Natl. Acad. Sci. U.S.A. 77:2819-2823). The most preferredmethod to generate genomic fragments is using restriction enzyme orphysical shearing. The genomic fragments are then ligated topromoterless reporter gene to generate gene fusion. The above methods togenerate fusions are well known in the art.

[0190] This invention also provides a method to use the genomeregistered collection of gene fusions in a liquid format to discovergene fusions useful as biosensors. For example, 39 gene fusions werefound to be upregulated when exposed to limonene for 135 minutes(Example 7). By use of these gene fusion in a strain to be used as theproduction organism for limonene, the presence of limonene, or theoptimum conditions for the limonene production can be measured by theupregulation of bioluminescence.

[0191] Gene expression profiles yield information relevant tounderstanding gene function and modes of chemical action. Likewise, suchinformation can be gained by analysis of genetic alterations resultingin loss of function, reduced levels, or over-expression of geneproducts. Thus, an “array of arrays” can be used to enhance both mode ofaction studies and functional genomics. Flow diagrams I and II depicttwo ways such arrays of arrays can be used.

[0192] Flow diagram I represents the several tests can be performed on agiven perturbation that changes the environment of the cell.

[0193] Flow diagram II describes that as data from arrays of arrays isanalyzed, altered responses of interest can be further analyzed byselected tools in the array of arrays.

[0194] These arrays of arrays can be built by generating largecollections of genome-registered mutations in genes of an organism.Several methods are available including but not limited to classicalradiation and chemical induced mutagenesis as well as more modemgenomic-based techniques including random in vivo transposition followedby sequencing of junctions to determine the gene disrupted, targeted invitro transposition into individual genes followed by homologousrecombination in vivo to generate disruptants, and primeroligonucleotide generated deletion insertion alleles generated in vitroby PCR and subsequently recombined into the genome. Spontaneous mutantscan also be selected by a variety of methods (LaRossa, R. A. (1996) InEscherichia coli and Salmonella: Cellular and Molecular Biology. ASMPress, p. 2527-2587.). Likewise, a large collection of genome-registeredgenetic alterations that result in over-expression can be generated.This can be accomplished in several ways including but not limited togenetic selection (LaRossa, R. A. (1996) In Escherichia coli andSalmonella: Cellular and Molecular Biology. ASM Press, p. 2527-2587),cloning on multicopy plasmids, placement of the gene to beover-expressed behind a strong promoter, and placement of the gene to beover-expressed behind a regulated promoter.

[0195] Perturbations are any alteration of environment or genotype.Perturbations that change the environment include but are not limited tophysical properties, such as radiation fluence, radiation spectrum,humidity, substratum, or temperature; nutritional properties, such ascarbon source, energy source, nitrogen source, phosphorus source, sulfursource, or trace element sources; biological properties, such aspresence of competitors, predators, commensals, pathogens such as phageand other viruses, the presence of toxins, or bacterocins; and chemicalproperties, such as presence of chelators, inhibitors, toxicants orabnormal levels of normal metabolites.

[0196] Several tests can be performed on a given perturbation thatchanges the environment of the cell (Flow Diagram I). Responses includepatterns of gene expression (e.g., reporter gene expression, presence orabsence of specific protein or intermediates) and phenotypic effects ofgenetic alterations; these responses can be analyzed concomitantly.Examples of phenotypes that may be screened for in the present methodinclude but are not limited to metabolic capacity (e.g., carbon sourcerequirement, auxotroph requirement, amino acid requirement, nitrogensource requirement, and purine requirement); Resistance to inorganicchemicals (e.g., acid, arsenate, azide, heavy metals, and peroxide);Resistance to organic and biological chemicals (e.g., antibiotics,Acridine, actinomycin, amino purine, amino phenylalanine, colicin,ethanol, fluoroacetate, mitomycin C, and nalidixic acid); Resistance tobiological agents (e.g., phages); Resistance to physical extremes (e.g.,temperature, pH, osmotolerance and radiation). The phenotypes amenableto detection by the present invention are numerous and a full review maybe found in, Robert LaRossa: Escherichia coli and Salmonella: Cellularand Molecular Biology (1996) ASM press p. 2527-2587). This will yield anenhanced empirical matching of one perturbation to another. For instanceif one chemical yields a very similar pattern of effects in all tests toanother chemical, then the likelihood of a similar mode of action of thetwo chemicals is high. Secondly, such concomitant analysis of severalpatterns will enhance understanding of gene function. For example, if agroup of genes is regulated similarly by environmental perturbation andgenetic perturbation (i.e., mutation) in this group of genes havesimilar phenotypic effects, then similar function can be hypothesized.

[0197] Flow Diagram II depicts that as data from arrays of arrays isanalyzed, altered responses of interest can be further analyzed byselected tools in the array of arrays. For example, one can evaluate anyperturbation by asking which mutants are hypersensitive orhyper-resistant to the environmental change. Look at the gene expressionprofile of wild type and the altered mutants in response to theenvironmental change and in its absence. Another approach to examinegenetic changes is to compare the genome-registered collection ofmutants to the wild type by examining how growth characteristics varybetween the mutants and wild type with changes in a wide range ofenvironmental parameters. Differences of interest are then followed upwith gene expression profiling.

[0198] As arrays of arrays are utilized, the massive amount of data onphenotypes, which results from interactions between genotypes and theenvironment and found by changing either the genetic composition or theculture conditions, will allow interpretation of the interplay betweenmutants and gene expression profiles. Analysis of such interactions willbe also useful for discovery of gene function and determining the modesof chemical action. Furthermore, these analyses may lead toidentification of useful targets for pharmaceuticals, antimicrobials, oragrochemicals, development of environmental diagnostic tests, ordevelopment of high throughput screen based on modes or sites ofchemical action.

EXAMPLES

[0199] The present invention is further defined in the followingExamples. It should be understood that these Examples, while indicatingpreferred embodiments of the invention, are given by way of illustrationonly. From the above discussion and these Examples, one skilled in theart can ascertain the essential characteristics of this invention, andwithout departing from the spirit and scope thereof, can make variouschanges and modifications of the invention to adapt it to various usageand conditions.

General Methods

[0200] Standard recombinant DNA and molecular cloning techniques used inthe Examples are well known in the art and are described by Sambrook,J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A LaboratoryManual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, (1989)(Maniatis) and by T. J. Silhavy, M. L. Bennan, and L. W. Enquist,Experiments with Gene Fusions, Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y. (1984) and by Ausubel, F. M. et al., CurrentProtocols in Molecular Biology, pub. by Greene Publishing Assoc. andWiley-Interscience (1987).

[0201] The meaning of abbreviations is as follows: “KB” meanskilobase(s) “hr” means hour(s), “min” means minute(s), “sec” meanssecond(s), “d” means day(s), “ml” means milliliter(s), “μl” meansmicroliter(s), “nl” means nanoliter(s), “μg” means microgram(s), “ng”means nanogram(s), “mM” means millimolar, “μM” means micromolar.

[0202] Media and Culture Conditions:

[0203] Materials and methods suitable for the maintenance and growth ofbacterial cultures were found in Experiments in Molecular Genetics(Jeffrey H. Miller), Cold spring Harbor Laboratory Press (1972), Manualof Methods for General Bacteriology (Phillip Gerhardt, R. G. E. Murray,Ralph N. Costilow, Eugene W. Nester, Willis A. Wood, Noel R. Krieg andG. Briggs Phillips, eds), pp. 210-213, American Society forMicrobiology, Washington, D.C. or Thomas D. Brock in Biotechnology: ATextbook of Industrial Microbiology, Second Edition (1989) SinauerAssociates, Inc., Sunderland Mass. All reagents and materials used forthe growth and maintenance of bacterial cells were obtained from AldrichChemicals (Milwaukee, Wis.), DIFCO Laboraoties (Detroit, Mich.),Gibco/BRL (Gaithersburg, Md.), or Sigma Chemical Company (St. Louis,Mo.) unless otherwise specified.

[0204] LB medium contains following per liter of medium: Bacto-tryptone(10 g), Bacto-yeast extract (5 g), and NaCl (10 g).

[0205] Vogel-Bonner medium contains the following per liter: 0.2 gMgSO₄.7H₂O, 2 g citric acid.1H₂O, 10 g K₂HPO₄ and 3.5 g NaHNH₄PO₄.4H₂O.

[0206] Minimal M9 medium contains following per liter of medium: Na₂HPO₄(6 g), KH₂PO₄ (3 g), NaCl (0.5 g), and NH₄Cl (1 g).

[0207] Above media were autoclaved for sterilization then 10 ml of 0.01M CaCl₂ and 1 ml of 1 M MgSO₄.7H₂O were added to M9 mediums. Carbonsource and other nutrient were added as mentioned in the examples. Alladditions were pre-sterilized before they were added to the media.

[0208] Molecular Biology Techniques:

[0209] Restriction enzyme digestions, ligations, transformations, andmethods for agarose gel electrophoresis were performed as described inSambrook, J., et al., Molecular Cloning: A Laboratory Manual, SecondEdition, Cold Spring Harbor Laboratory Press (1989). Polymerase ChainReactions (PCR) techniques were found in White, B., PCR Protocols:Current Methods and Applications, Volume 15(1993) Humana Press Inc.

Example 1 Construction, Sequencing and Registering a Random Library ofE. coli Genomic Fragments Fused to a luxCDABE Reporter

[0210] The random library of E. coli genomic fragments in plasmidpDEW201, which contains the origin of replication and bla from pBR322,four transcription terminators upstream of the promoterless P.luminescens luxCDABE genes, and a multiple cloning site that liesbetween the terminators and luxCDABE were constructed as previouslydescribed (Van Dyk et al., (1998) J. Bacteriol. 180:785-792; Van Dyk andLaRossa (1998) Methods in Molecular Biology: Bioluminescence, Methodsand Protocols, Humana Press Inc. vol. 102:85-95).

[0211] Briefly, chromosomal DNA isolated from E. coli strain W3110(Ernsting et al., (1992) J. Bacteriol. 174:1109-1118) was partiallydigested with the restriction enzyme Sau3A1, size fractionated byagarose gel electrophoreses, and a fraction with an average size ofapproximately 1.8 KB isolated. This fraction was ligated to pDEW201 thathad previously been digested with BamHI and treated with calf intestinalalkaline phosphatase. The ligation products were used to transformultracompetent E. coli XL2Blue cells (Stratagene) to ampicillinresistance using the protocol provided by Stratagene. Preliminarycharacterization of individual XL2Blue transformants that were picked inrandom indicated that a large percentage (16 of 16) contained insert DNAwith sizes ranging from 0.9 to 3.0 KB. Approximately 24,000 of thesetransformants were pooled and used as a source of heterogeneous plasmidDNA isolated using Qiagen tip20 columns (Qiagen Corp). This plasmid DNApool was used to transform E. coli DPD1675 (Nishimura et al. (1990).Nucleic Acids Res. 18:6169; Van Dyk and LaRossa (1998) Methods inMolecular Biology: Bioluminescence Methods and Protocols, Humana Pressinc. vol. 102:85-95) selecting for ampicillin resistance and using a 30minute phenotypic expression time to minimize the presence of siblings.A total of 8066 individual transformants were used to inoculate the96-wells of sterile Microtest III™ Tissue Culture Plates (Falcon®) eachcontaining 190 μl of Vogel-Bonner medium (Davis et al., (1980) Advancedbacterial genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y.) with glucose as a carbon source and supplemented with thiamine,uracil, proline, and 25 μg/ml of ampicillin. These plates were coveredand incubated overnight without shaking at 37° C. The overnight culturesin 96-well plates were used for permanent cryogenic storage in duplicateat −80° C. (Menzel, R., (1989) Anal. Biochem. 181:40-50). These 8066individual cultures are called the Lux-A Collection.

[0212] For DNA sequence analysis, one of the duplicate sets of the Lux-ACollection was thawed and used as an inoculum for cultures grown tosaturation in Terrific Broth (Gibco-BRL, Inc) containing 100 μg/mlampicillin in 96 well deep-well plates. Plasmid DNA was extracted fromthe cultures using the Qiagen R.E.A.L.™ prep method with the followingmodification: after lysis of cells, the plates were placed in a boilingwater bath for 5 minutes and then rapidly chilled in an ice-water bathbefore precipitation with Buffer 3. This modification preventeddegradation of the plasmid DNA by the nucleases present in the non-endAhost strain, DPD1675. DNA sequencing reactions were performed withapproximately 1 μg of plasmid DNA under standard ABI Prism™DyeTerminator Reaction Ready conditions with the primers pDEW201.forward(SEQ ID NO: 1, 5′-GGATCGGAATTCCCGGGGAT-3′) and pDEW201.reverse (SEQ IDNO:2, 5′-CTGGCCGTTAATAATGAATG-3′) to obtain sequence information fromeach end of the insert. DNA sequences were determined on ABI377™-XL96-lane upgraded Sequencers under 4× run conditions on 5% PAG(polyacrylamide gel) LongRanger™ (FMC, Inc.) gels and analyzed with ABIsoftware. DNA sequences were transferred to a UNIX based utility forfurther analysis. A homology search for the sequence from the beginningand end of each Lux-A clone (in both orientations) was performed againstthe complete E. coli sequence (Genbank accession U00096) using Pearson'sFASTA program (fasta3, Version 3.1t13). The essential Fasta options were−nQH −m 10 −z 0.

[0213] The essential data about each highly significant alignment (FASTAscore >1000, minimum overlap length >200, and minimum identity >70%) wasstored in a relational database (Sybase System 11, Sybase Inc.).

[0214] The location of Lux-A clone on the E. coli genome was then basedon the above computed homologies for both the beginning and end of theinsert, using the following rules:

[0215] i) Both distal and proximal ends of the insert must have anunambiguously high sequence homology with E. coli,

[0216] ii) the relative location and orientation of the matches of thesequence determined from the beginning and end of the clone implied areasonable length for the clone, which were known to fall in a fairlynarrow distribution.

[0217] In many cases the above procedure gave a single probablelocation, but in others there were multiple possible locations. Theresults were stored in the relational database.

[0218] A table of open reading frame annotations for E. coli wasdownloaded using NCBI's Entrez facility, and this data was stored in therelational database.

[0219] A web based, tabular interface to the data was created. Thisallows one to see the Lux-A clones in relation to the functionalannotation. A Java based graphical interface was also created to makepositional and directional relationships easy to visualize. Thus theLux-A Collection was registered to the E. coli genome.

Example 2 Validation of Lux-A Collection by Verification of SelectedGlobal Regulatory Responses

[0220] With the sequencing of Lux-A Collections and registering of themajority of the LuxA members, it was possible to further examine thebiological responses of strains containing luxCDABE fusions to othermembers of well-characterized regulatory circuits. Gene fusions to thelac operon, and members of the heat shock, SOS, SoxRS, and OxyR regulonswere selected from the Lux-A Collection and the responses to knowninducers of each of these global regulatory circuits were tested.

[0221] Growth Media and Chemicals.

[0222] A rich liquid medium, LB (Miller, J. H., (1972) Experiments inmolecular genetics. Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.), was used. When specified, ampicillin (Amp) was added at150 μg/ml. Ethanol (200 proof, Quantum Chemicals) was diluted directlyinto LB medium. A stock solution of 20 mg/ml nalidixic acid (SigmaChemical Co.) in 1 M NaOH was further diluted into LB medium. Likewise,a stock solution of 100 mg/ml methyl viologen (Sigma Chemical Co.) inwater was further diluted into LB medium. A 30% solution of hydrogenperoxide (EM Science) was diluted into LB medium to the desiredconcentrations.

[0223] Re-Isolation from Microplates, Growth of Cultures, and TestingStress Responses.

[0224] Selected strains were reisolated from the Lux-A Collectionmicroplates stored at −80° C. by streaking for single colonies on LB-Ampplates. The purity of the cultures in the wells was tested byinoculating four single colonies into 100 μl of LB medium in a 96 wellluminometer plate (Microlite, Dynex Technologies). Consistentbioluminescence of each set of four isolates provided evidence of clonalpurity.

[0225] To test the stress responses, one or two single colonies of eachselected strain were used to inoculate 5.0 ml of LB medium containing150 μg/ml of ampicillin. These cultures were grown overnight at 37° C.then diluted by adding 50 μl or 100 μl of the overnight culture into10.0 ml of fresh LB medium (without ampicillin) and grown with agitationat 37° C. until the culture was in exponential phase with readings on aKlett-Summerson colorimeter containing the red filter of between 10 and40. These actively growing cultures were immediately used to initiate astress response experiment by adding 50 μl of cultures to 50 μl of LBmedium containing various concentrations of chemical in the wells a 96well luminometer plate. This division of the actively growing culture atthe time of chemical addition ensured identical populations were presentwhen the stress was imposed. Light production was measured in a DynexML3000 luminometer at 37° C. The dimensionless units of lightproduction, relative light units (RLU), are obtained by comparison withthe light reading from an internal light-emitting diode. The cycle modeof the ML3000 luminometer, similar to previous descriptions (Van Dyk etal., Appl. Environ. Microbiol. 60:1414-1420) was used.

[0226] Gene Fusions to the lacZYA Operon.

[0227] Transcription of the very well characterized lac operon isregulated by both specific and global regulatory circuits. Specific,negative regulation is mediated by the lacI-encoded repressor (Choy andAdhya, (1996) In Escherichia coli and Salmonella: Cellular and MolecularBiology. ASM Press pp 1287-1299). Global regulation in response toglucose availability is mediated by positive transcriptional activationof cAMP-CRP (Botsford and Harman (1992) Cyclic AMP in prokaryotes.Microbiol. Rev. 56:100-122). The Lux-A Collection contains three membersthat are fusions of luxCDABE to the lac operon as shown in FIG. 1. Totest for the appropriate response of these gene fusions to glucose, eachwas reisolated and tested for bioluminescence when grown in the presenceor absence of glucose in LB medium. The results are shown in Table 2.TABLE 2 Fusions to the lac operon in the Lux-A Collection. RLU of 100 μlovernight cultures Lux Clone LBAmp¹⁵⁰ + 0.4% glucose LBAmp¹⁵⁰lux-a.pk034.a6 (a) 0.002 23.0 lux-a.pk034.a6 (b) 0.001 18.8lux-a.pk050.b9 (a) 0.001 26.0 lux-a.pk050.b9 (b) 0.001 0.007lux-a.pk065.d4 (a) 0.001 19.1 lux-a.pk065.d4 (a) 0.001 19.6

[0228] Two isolated single colonies of each culture were tested bygrowing overnight in the specified medium then measuring thebioluminescence of 100 μl in an ML3000 luminometer. LBAmp medium is LBmedium containing ampicillin at 150 mg/ml concentration.

[0229] With one exception, each of the isolated colonies from thecultures in the Lux-A Collection was much more highly bioluminescent inthe absence of glucose than in its presence. One colony from thelux-a.pk050.b9 that was not highly bioluminescent might be due tocontamination of the culture in that well. Other putative crosscontamination events have not been observed. Each of the lacZ-luxCDABEfusion strains had at least one reisolated colony that gave the expectedresponse to glucose. Thus, the appropriate biological response of theseLux-A Collection members was verified.

[0230] Heat Shock Regulon Gene Fusions.

[0231] The Lux-A Collection was examined to find fusions to genes in theσ³²-controlled heat shock regulon (Gross, C. A., (1996) In Escherichiacoli and Salmonella: Cellular and Molecular Biology, Second ed. ASMPress, pp. 1382-1399). Table 3 shows the genes or operons for which theLux-A Collection was searched and the number of gene fusions found foreach gene. TABLE 3 Lux-A Collection Gene Fusions to Heat Shock Regulonmembers Number of Heat shock gene Fusions in the or operon Lux-ACollection grpE 0 lon 1 clpPX 0 dnaKJ 0 groESL (mopAB) 0 rpoD 3 htpG 0clpB 1 htpX 0 htgA (htpY) 0 hslT (ibpA) 0 rfaDFCL (htrM) 1

[0232] While there was not complete representation of the each member ofthe heat shock regulon, lux fusions to four of twelve heat shockpromoter (33%) were found. Thus, the Lux-A Collection contains membersthat should report on activation of this stress response.

[0233] In addition, strains with the previously constructedgrpE-luxCDABE gene fusion in parental plasmid pDEW201 (Van Dyk et al.,(1998) J. Bacteriol. 180:785-792) had been placed in selected wells ofthe Lux-A Collection plates as a control. One of these was also selectedfor testing. Table 4 summarizes the information on the six strains thatwere reisolated from wells of the frozen Lux-A Collection. TABLE 4 Heatshock regulon gene fusions Genes in 3% Ethanol Strain cloned BasalResponse Lux clone name insert size fusion to: DNA RLU* ratio# lux-a.pk057.h1 DPD2241 581 grpE yfjB' grpE' 61.1 5.2 lux-a. DPD2242 2154 lon‘clpP clpX 6.71 5.6 pk021.g11 lon’¹ lux-a. pk089.e7 DPD2246 1989 rpoD‘dnaG 9.49 2.4 rpoD’² lux-a. pk054.c3 DPD2243 2319 clpB ‘sfhB yfiH 38.54.7 clpB’³ lux-a. pk040.e4 DPD2244 2650 rfaDFCL yibB' rfaD 33.1 1.4rfaF'

[0234] The light production from each of these strains in the absence ofstress is consistent with each of these chromosomal DNA fragmentscontaining an active promoter because these values are much greater thanthat from a culture carrying the parental plasmid pDEW201, which had0.002 RLU under the same conditions (Van Dyk et al., (1998) J.Bacteriol. 180:785-792). These strains were tested for their ability toreport on the heat shock stress response by using 5, 4, 3, and 2 (v/v) %ethanol, a known, potent chemical inducer. For each of the six strains,at least two concentrations of ethanol resulted in increasedbioluminescence as compared with the control, untreated culture. Table 4contains the response ratio for each strain to 3% ethanol at 40 minutesof treatment. These data demonstrate that these heat shock regulon genefusions from the Lux-A Collection report the induction of the heat shockresponse.

[0235] SOS Regulon Gene Fusions.

[0236] In a like fashion as described above, the Lux-A Collection wasexamined to find fusions to genes in the SOS DNA damage response regulon(Kim et al., (1997) Proc Natl Acad Sci USA 94(25):13792-7; Walker, G. C.(1996) In Escherichia coli and Salmonella: Cellular and MolecularBiology. ASM Press pp. 1400-1416). Table 5 shows the genes or operonsfor which the randomly generated Lux-A Collection was examined and thenumber of gene fusions present for each gene. TABLE 5 Quantities ofLux-A Collection Gene Fusions to SOS Regulon members Number of SOS geneFusions in the or operon Lux-A Collection umuDC 0 recA 2 uvrA 1 uvrB 0sulA (sfiA) 0 uvrD 1 recN 0 polB (din A) 0 dinP (dinB) 1 dinD 0 dinF 1dinG 1 dinI 0 ruvAB 1

[0237] Of the fourteen SOS regulon genes or operons examined, seven(50%) were found to be represented by useful fusions in the Lux-ACollection. Thus, this collection contains members that should report onactivation of the SOS stress response. Table 6 summarizes theinformation on the eight strains that were reisolated from wells of thefrozen Lux-A Collection. TABLE 6 SOS regulon gene fusions Nalidixic acidStrain Genes in Insert Basal response Lux clone name insert size fusionto: DNA RLU* ratio# lux-a. DPD2247 2840 dinF plsB(−)' dgkA(+) 0.224 2.2pk085.a5 lexA(+) dinF(+)' lux-a. DPD2248 2022 dinG ‘rhlE(+) ybiA(−) 32.82.0 pk0024.f5 dinG(+)’ lux-a. DPD2249 2125 dinP fhiA(−)'mbhA(+) 4.41 5.0pk055.a3 dinP(+)' lux-a. DPD2250 1789 recA ‘mtlB(−) ygaD(−) 97.5 1.8pk022.d4 recA(−)’ lux-a. DPD2251 1782 recA ‘mtlB(−) ygaD(−) 99.5 2.8pk085.b5 recA(−)’ lux-a. DPD2253 2105 uvrA yjcC(+)' yjcB(−) not 1.4pk01.b6 ssb(+) uvrA(−)' done lux-a. DPD2254 1632 uvrD ‘xerC(+) 15.7 1.2pk052.b6 yigB(+) uvrD(+)’ lux-a. DPD2293 1890 ruvA(−) ‘yebC(−) ruvC(−)14.3 4.6 pk058.f2 yebB(+) ruvA(−)’

[0238] Each of these strains that were tested for basal bioluminescencein defined medium had a greater level of light production than did astrain containing the parental plasmid, indicating the presence of apromoter driving expression of the luxCDABE reporter. The ability ofthese promoter-luxCDABE fusions to report activation of the SOS stressresponse was tested using nalidixic acid, a known, potent chemicalinducer. The final concentrations of nalidixic acid were 1, 5, 25, and125 μg/ml for all but strain DPD2293, which was tested at 80, 40, 20,10, 5, 2.5, and 1.25 μg/ml. For each of the eight strains, at leastthree concentrations of nalidixic resulted in increased bioluminescence.Table 6 gives the response ratio to 5 μg/ml of nalidixic acid at 110minutes after addition. These data demonstrate that induction of the SOSresponse is reported by increased bioluminescence for these SOS regulongene fusion in the Lux-A Collection.

[0239] Specificity of responses was tested by measuring the effect ofethanol on these SOS regulon gene fusions. In each case there was littleto no increased bioluminescence induced by 5, 4, or 3% (v/v) ethanoltreatment. For example, the treatment with 3% ethanol at 40 minutesafter addition, a condition which resulted in increased light productionfrom all the heat shock regulon gene fusions (Table 4), for strainDPD2249 containing a dinP-luxCDABE gene fusion yielded a response ratiowas 0.80. Similarly, when strain DPD2243 containing a heat shock regulonclpB-luxCDABE fusion was tested with nalidixic acid, no increase inbioluminescence was observed; at 110 minutes after addition of 5 μg/mlnalidixic acid, the response ratio was 0.40. Thus, the specificity ofthe heat shock response to ethanol and the SOS response to nalidixicacid was shown.

[0240] SoxS-Regulated Oxidative Damage Regulon Gene Fusions.

[0241] The Lux-A Collection was also examined for the presence offusions to genes in the SoxR and SoxS regulated oxidative stressresponse regulon (Koh et al., (1999) Mol. Gen. Genet. 261:374-380;Rosner and Storz (1997) Curr. Top. Cell. Regul. 35:163-177; Van Dyk etal., (1998) J. Bacteriol. 180:785-792). Table 7 below shows results.TABLE 7 Quantities of Lux-A Collection Gene Fusions to the SoxR/Sregulon members Number of SoxR/A regulon Fusions in the gene or operonLux-A Collection sodA 0 nfo 0 fumC 0 achA 0 fpr 0 zwf 3 micF 0 acrAB 0inaA 1 pqiAB 0 ribA 1 poxB 1

[0242] In a similar fashion to the heat shock and SOS regulons, theSoxR/S regulon was not fully represented in the Lux-A Collection.Nevertheless, the collection contained gene fusions to 33% of the SoxR/Sregulon gene or operons that would be expected to report on theactivation of this stress response. Table 8 summarizes the informationon the six strains that were reisolated from wells of the frozen Lux-ACollection. TABLE 8 SoxR/S regulon gene fusions Methyl viologen StrainGenes in Insert Basal response Lux clone name insert size fusion to: DNARLU* ratio lux-a. DPD2278 1708 zwf pykA(+)’ 5.0 Not tested pk053.b1yebK(+) zwf'(−)' lux-a. DPD2272 1708 zwf pykA(+)’ 16.9 3.6 pk082.g7yebK(+) zwf'(−)' lux-a. DPD2279 1709 zwf pykA(+)’ 4.2 Not testedpk088.e1 yebK(+) zwf'(−) lux-a. DPD2286 2308 ribA yciM(+)' 5.8 4.7pk078.d2 o102(+) pgpB(+) ribA(−)' lux-a. DPD2087 1583 inaA 'glpQ yhaH9.8 9.0 pk014.a9 inaA’ lux-a. DPD3509 1131 poxB ‘b0872 poxB’ 1.5 15.0 pk071.a11

[0243] Like the SOS and heat shock regulon fusions, these had basallight production greater than the promoterless parental plasmidindicating the presence of a promoter. Responsiveness of four of thesestrains to a known inducer of the SoxR/S regulon, methyl viologen, wastested at 1000, 500, 250, 125, 62, 31 and 16 μg/ml. Each of these sevenmethyl viologen concentrations induced increased bioluminescence fromeach of the four strains. Table 8 gives the response ratio for 250 μg/mlat 120 minutes of treatment for the four tested strains. Here again, thebiologically appropriate response was observed.

[0244] OxyR-Regulated Oxidative Damage Regulon Gene Fusions.

[0245] Fusions to OxyR regulated genes (Rosner and Storz. (1997) Curr.Top. Cell. Regul. 35:163-177) were found in the Lux-A Collection assummarized in Table 9. TABLE 9 Quantities of Lux-A Collection GeneFusions to the OxyR regulon members Number of OxyR regulon Fusions inthe gene or operon Lux-A Collection katG 0 gorA 0 dps 0 ahpCF 3

[0246] Of these four genes or operons controlled by OxyR, fusions to one(25%) was available in the Lux-A Collection. Table 10 summarizes theinformation on the three strains, each containing a fusion of the ahpCFoperon regulatory region to the luxCDABE reporter, that were reisolatedfrom wells of the frozen Lux-A Collection. TABLE 10 OxyR regulon genefusions H₂O₂ response Genes in RLU H₂O₂ ratio in insert fusion Insertinitial response pcnB⁻ Lux clone Strain name size to: DNA screen* ratio#host‡ lux-a. DPD2283 1184 ahpC dsbG' 92.2 1.3 16.5 pk051.d5 ahpC(+)'lux-a. DPD2284 1184 ahpC dsbG' 68.3 1.6 15.3 pk051.e3 ahpC(+)' lux-a.DPD2285 1182 ahpC dsbG' 47.2 1.2 not done pk03.d6 ahpC(+)'

[0247] The high level of light production from these three strainscontaining ahpC-luxCDABE gene fusions indicates that they each containeda very active promoter. To test if these strains would report onactivation of the OxyR-controlled oxidative stress response, each wastreated with hydrogen peroxide at 0.016, 0.008, 0.004, 0.002, 0.001,0.0005, 0.00025%. At best, the bioluminescence was minimally induced.Table 10 shows the response ratio to treatment with 0.002% hydrogenperoxide at 30 minutes after treatment.

[0248] Previously, another luxCDABE fusion in plasmid pDEW201 to a genein the OxyR regulon, katG had been shown to yield larger response ratiosto hydrogen peroxide when the plasmid was moved to an E. coli hoststrain containing a pcnB mutation (Van Dyk et al., (2000) in press. InA. Mulchandani and O. A. Sadik (ed.), Recent Advances in EnvironmentalChemical Sensors and Biosensors. ACS Symposium Series). Mutations inthis gene result in reduced plasmid copy number for plasmids withorigins of replication like that in pBR322 (Lopilato et al., (1986) Mol.Gen. Genet. 205:285-290), thus resulting in reduced basal expression ofgene fusions carried on such plasmids. Accordingly, to test if reducingthe copy number of the ahpC-luxCDABE fusions would also yield morereliable detection of the OxyR-mediated stress response, plasmid DNAtaken from two of these Lux-A Collection strains was moved bytransformation into a pcnB³¹ mutant host strain. The two resultingstrains were tested for responses to hydrogen peroxide at 0.004, 0.002,0.001, 0.0005, 0.00025, 0.00012, 0.00006%. Each of these sevenconcentrations dramatically induced increased bioluminescence from bothahpC-luxCDABE fusion strains in the pcnB³¹ host. The last column ofTable 10 gives the response ratio to treatment with 0.002% hydrogenperoxide at 30 minutes. Thus, the appropriate biological response fromthese fusions to a highly expressed gene was obtained when the copynumber of the gene fusion was reduced.

[0249] In one instance where the response from the plasmid-borne genefusion to a highly expressed gene was weak, it was demonstrated thatreduction of plasmid copy number with a pcnB mutation resulted in morepotent induction. Other fusions to highly expressed genes can also bemoved to a host strain with reduced copy number, such a pcnB mutant, orintegrated into the chromosome at a gene dosage of one (Elsemore, D. A.(1998) Methods in Molecular Biology: Bioluminescencent Protocols., vol.102, p:97-104, Humana Press, Inc).

Example 3 Use of a Genome-Registered Collection of Reporter Gene Fusionsto Confirm or Question Results from DNA Array Analysis and to DevelopHigh throughput Screens Based on Gene Expression

[0250] DNA Microarray Experiment with Mitomycin C (MMC)

[0251] The E. coli strain MG1655 (rph-1) was used. Cultures grown in LBat 37° C. overnight were diluted 1 to 250 into fresh LB and grown at 37°C. with aeration. Each subculture was split into two 100 ml cultureswhen the reading on a Klett-Sammerson colorimeter with the red filterreads 20 Klett units. MMC (Sigma, dissolved in ddH₂O) at a finalconcentration of 250 ng/ml, a sub-lethal dose, was added to one of thesplit cultures. The other culture was a no addition control. Incubationat 37° C. continued for another 40 minutes, then cells were collectedfor preparing total RNA. The MMC treated culture and its control reached60 and 55 Klett units, respectively after 40 minute treatment.

[0252] Total RNA purification, first-strand cDNA labeling, preparationof the E. coli whole genome high-density microarray chips, hybridizationand data analysis were done as previously described (Wei et al. (2001)J. Bacteriol. 183: 545-556). Both cy3 and cy5 were used in probelabeling, and the hybridization experiments were repeated by swappingthe fluorescence cy dyes between each pair of MMC treated sample and itsblank control.

[0253] Ratios of expression in the mitomycin C treated samples vs.controls were calculated for all genes in the DNA array. Ratios greaterthan or equal to 2 were considered induced genes, while those withratios less than 2 fold were considered uninduced. The known SOS genes(Kim et al., (1997) Proc Natl Acad Sci USA 1997 Dec 9;94(25):13792-7;Lomba et al., (1997) FEMS Microbiol Lett 156:119-122; Walker, G. C.(1996). In Escherichia coli and Salmonella: Cellular and MolecularBiology. ASM Press pp.1400-1416) fell into both the induced (Table 11)and uninduced classes (Table 12). In addition, 20 genes not previouslyknown to be induced by MMC were observed to be induced in the arrayexperiment (Table 13). TABLE 11 Known SOS genes induced by MMC in arrayexperiment Fold induction/ Available Lux Induction Gene array experimentFusion of Fusion recN 8.3 NO recA 3.0 YES YES lexA 3.6 NO dinI 6.7 NOdinD 2.2 NO uvrA 2.3 YES YES uvrB 2.1 NO ruvA 2.0 YES YES sulA 5.8 NOumuC 2.1 NO dinB (dinP) 2.0 YES YES b1848 (yebG) 5.8 NO

[0254] TABLE 12 Known SOS genes NOT induced by MMC in array experimentAvailable Fold induction/ Lux Induction Gene array experiment Fusion ofFusion uvrD 1.4 YES YES polB (dinA) 1.2 NO dinG 1.4 YES YES dinF 1.8 YESYES himA 1.3 NO ruvB 1.5 NO umuD 1.2 NO

[0255] TABLE 13 Genes not previously known to be DNA damage-induciblefound induced by MMC in array experiment Available Fold induction/ LuxInduction of Gene array expt Fusion Fusion mioC 2.1 NO xseA 2.0 NOinsB_2 2.2 NO insB_1 2.1 NO insA_4 2.1 NO secG 2.2 NO exbD 2.2 YES NOtrkH 2.1 YES NO infA 2.3 NO hslS 6.7 NO hslT 4.0 NO cspA 2.9 NO dniR 2.1YES NO b0531 3.3 NO b1847 (yebF) 3.3 YES YES (when inverted) b1228 2.5NO b2940 2.3 NO b0571 (ylcA) 2.2 YES NO b2559 2.0 NO b3199 2.0 NO

[0256] Verification of Expected Responses of MMC Induction of Known SOSGenes

[0257] The Lux-A Collection of reporter gene fusions was examined forpresence of fusions to the genes in Table 11. Four were found to bepresent in the collection. Each of these was tested for induction by MMCat several doses over a time course of 100 minutes. The expectedinduction response of a lag period with no change in gene expressionfollowed by an induction of increased bioluminescence was observed inall four cases at several doses of MMC. Thus, this demonstrates thatresults from DNA array experiments can be verified by usingcorresponding gene fusions.

[0258] Questioning the Negative Result of Non-Induction of Known SOSGenes

[0259] As expected, the expression of the known SOS genes was elevated;however the expression of several was elevated less than 2-fold (Table12) and as such were within a large group of 792 genes the expression ofwhich was elevated by 20% or more. Most of these are likely due toartifacts in the array data rather than to actual biologically relevantresponses. To test if strains carrying luxCDABE gene fusions would yieldthe expected positive result, the three gene fusions that were availablein the Lux-A Collection of reporter gene fusions were tested formitomycin C responses. In all three cases, mitomycin C induced increasedbioluminescence. Thus, this demonstrates that negative results from DNAarrays can be questioned by contradictory positive results withcorresponding gene fusions.

[0260] Questioning or Confirmation of Induction of Previously UnknownMMC Inducible Genes

[0261] The genes that were not previously known to be induced withmitomycin C were further examined for correlation of DNA array and genefusion experimental data. For this class of genes, four fusions wereavailable in the Lux-A Collection of reporter gene fusions.

[0262] The corresponding luxCDABE fusions to these four genes providedno evidence of increased gene expression induced by MMC.

[0263] An additional gene fusion in the Lux-A Collection of gene fusionswas found to have a genomic fragment that when inverted would result ina fusion to yebF. In this case, a divergent promoter to the purT genewas present in the chromosomal fragment and strains containing thebackward yebF fusion produced light. Following isolation of plasmid DNA,XmaI digestion that releases the insert DNA from the vector, religationand transformation, 10% of the transformants produced light. Of these,20% (or 2% of the initial transformants) were found to be highly inducedby nalidixic acid, another DNA damaging agent. This result stronglysuggested that inversion of the insert DNA had occurred in thesenalidixic-acid inducible gene fusions. The orientation of the insertedsegment to yield a fusion of yebF to the luxCDABE operon was confirmedby DNA sequence analysis. Furthermore, induction by mitomycin C wasdemonstrated, as shown in FIG. 2.

Example 4 Functional Definition of Postulated Promoter Regions

[0264] The genes encoding production of type I extracellularpolysaccharide in E. coli are located in a cluster of 20 genes(Stevenson et al. (1996) J. Bacteriol. 178: 4885-4893). An upstreampromoter for these genes has been identified (Stout, V. (1996) J.Bacteriol. 178: 4273-4280) and its regulation has been characterized(Gottesman, S. (1995) Two-component Signal Transduction. AmericanSociety of Microbiology pp253-262; Wehland and Bernhard (2000) J. Biol.Chem. 275:7013-7020). Nonetheless, the transcriptional organization ofthis region has not been completely defined. The annotated sequence forthese genes (Blattner et al. (1997) Science 277:1453-1462), which aretranscribed from one strand of the genome, suggests the existence ofseveral putative promoters and activator binding sites. Furthermore, aprediction of operon structure in this region suggests that these genesmay be organized into several transcriptional units (Thieffry et al.(1998) Bioinformatics 14:391-400). FIG. 8 summarizes the predictedpromoters in this region. Unexpectedly, an E. coli DNA microarray-basedexperiment with strains 397C, containing a truncated β' subunit of RNApolymerase, and P90, an isogenic rpoC⁺ control, suggested that RNAtranscripts in this region were affected by the rpoC mutation.Expression of genes b2043 through b2062 was coordinately upregulated inthe rpoC mutant (FIG. 8). The elevated expression is most readilyexplained if a single transcript starts before b2062 and ends betweenb2043 and b2042 (galF). If this is true, the region upstream of b2062should contain a promoter. That region was fused to the luxCDABE operonin lux-a.pk033.g2. Transformants of P90 and 397C were grown at 30° C. inLB medium containing 100 μg/ml ampicillin. Bioluminescence andturbidity, recorded with a Klett-Summerson colorimeter (Van Dyk et al.(1995) J. Bacteriol. 177:6001-6004), of actively growing cultures wereused for calculation of light production per 10⁹ cells. The unpairedt-test was used to compare quadruplicate bioluminescence measurements ofstrains carrying gene fusions to the control strain carrying theparental plasmid. The bioluminescence produced when the lux-a.pk033.g2gene fusion was placed into strain P90 was weak (0.56+/−0.06 RLU/10⁹CFU), but yet was significantly greater (P<0.0001) than thebioluminescence produced by the parental plasmid in the same host strain(0.028+/−0.010 RLU/10⁹ CFU). These data are consistent with a promoterin the region upstream of b2062 that is not very active in strain P90growing in LB medium. The bioluminescence of this gene fusion waselevated 1500-fold when placed in strain 397C (FIG. 8). This strongpromoter activity driving luxCDABE gene expression is, therefore,dependent upon the rpoC⁻ mutation. In contrast, several other genefusions to chromosomal DNA segments in this region, whether theycontained predicted promoter regions or not, had very low levels ofactivity in both the wild type and rpoC⁻ mutant (FIG. 8). Thus, the datafrom both the DNA microarray experiments and from gene fusions areconsistent with cotranscription of the twenty genes in this region. Theend of the operon was defined by a gene fusion to the galF upstreamregion (in lux-a.pk07.d12) that strongly drove luxCDABE transcription.The activity of this promoter region was not dramatically effected bythe rpoC mutation (FIG. 8), in agreement with the DNA array data.

Example 5 Highly Parallel Transcription Analysis Using a High DensityArray of Cellular Reporters

[0265] Proof of principle with Luxarray 0.5

[0266] A solid phase assay system consisting of reporter clones growingin the presence or absence of a perturbation, biological, environmentalor chemical was developed. This was accomplished by growing thereporters on a porous membrane (Biodyne B, Nunc) seated on top of solidgrowth media in a culture dish (OmniTray, Nunc). Luminescence wasmeasured either by a luminometer or by creating an image of the entireculture and quantitating the pixel density. Effects of perturbants canbe determined by comparison of luminescence generated by treated andcontrol cultures. Growth on the surface of the membrane allows thereporter assay to be moved between conditions as required. Experimentalprotocols often involve perturbations that prohibit long term exposuredue to cell death or other irreversible effects. The ability to move theentire array to new growth conditions allows one a great variety ofexperimental schemes such as pulsed or pulse/chase exposures,reversibility, and short term kinetic studies.

[0267] Several important characteristics of the assay system needed tobe evaluated. In particular, growth density and conditions, sensitivity,reproducibility, and the ability to perturb the reporters and detectchanges were major focus points. Development of this assay system wasinitiated using a collection of 10 well characterized clones, theparental plasmid clone, and media alone (Table 14). The set of 10 clonesrepresented a distribution of signal strength as well as response toperturbation with DNA damaging agents, in particular nalidixic acid.Initial experiments were designed to benefit from the sensitivity andsimplicity of measuring luminescence with a 96-well luminometer (DynexML3000). Printing was accomplished using a BioMek 2000 (Beckman Coulter)equipped with a High Density Replication Tool (HDRT). Sterilization inbetween transfers was accomplished by soaking the pins successively in0.2% SDS in water, sterile water, and 70% ethanol. After sterilization,the pins are air-dried prior to the next transfer. TABLE 14 CloneBiolumi- Strain No. Fusion to: nescence* Comment DPD2083 N.A. N.A. noneParental plasmid without insert DPD2282 65.d4 lacZ moderate“Constitutive” expression in LB/ low expression in LB + glucose DPD224785.a5 dinF low SOS regulon/ Nalidixic acid and Mitomycin C inducibleDPD2248 24.f5 dinG moderate SOS regulon/ Nalidixic acid and Mitomycin Cinducible DPD2249 55.a3 dinP moderate SOS regulon/ Nalidixic acid andMitomycin C inducible DPD2250 22.d4 recA high SOS regulon/ Nalidixicacid and Mitomycin C inducible DPD2253 01.b6 uvrA moderate SOS regulon/Nalidixic acid and Mitomycin C inducible DPD2242 21.g11 lon moderateHeat shock regulon DPD2243 54.c3 clpB moderate Heat shock regulonDPD2245 42.c8 rpoD low Heat shock regulon DPD2084 06.b4 yciG lowSigmaS-dependent stress responsive DPD2090 23.c7 osmY lowSigmaS-dependent stress responsive

[0268] Strains were grown overnight at 37° C. in 40 μl LB in 96-welldishes. These cultures were designated “printing plates” and were usedas the cell source to manufacture the arrays.

[0269] Membranes were sterilized with UV illumination for 10 min thenplaced in contact with prewarmed media in a culture dish. The clones andcontrols were printed onto an approximately 8×12 cm membrane mimickingthe pattern of a 96-well plate (FIG. 3A). Strains were printed togenerate 8 repeating sections of the 10 strains and two controls andplaced in a Dynex ML3000 luminometer prewarmed to 37° C. Luminescencedata was collected for each spot for 40 cycles of 20 min duration(overnight). FIG. 3B shows the signal collected for each clone as afunction of time. FIG. 3C shows signal collected from the replicatespots of the same clone (recA-luxCDABE). The data clearly indicated thatthe strains were well behaved in the new solid phase system. Withoutexception, the signal strength measured corroborated the data obtainedin liquid culture. Additionally, replicates varied minimally, againsimilar to liquid culture measurements.

[0270] Next the system was evaluated for its ability to determineresponses to perturbation, in this case, DNA damage caused by nalidixicacid. Parental clone and two nonresponding reporters, osmY, and lacZYA,and three DNA damage responsive reporters, uvrA, recA, dinG, werechosen. Strains were printed onto duplicate membranes over LB agar fromfresh overnight cultures as described and incubated for 6 hr at 37° C.to allow the cells to enter an exponential growth phase. The membraneswere moved to new, pre-warmed plates containing varying amounts ofnalidixic acid and light readings collected with an ML3000 luminometer.Luxarray 0.5 clones (FIGS. 4A and B) were printed onto membranes andgrown initially on LB then moved to plates containing 0 μg/ml(diamonds), 1 μg/ml (squares), or 5 μg/ml nalidixic acid (triangles).Luminescence was measured with a Dynex ML3000 luminometer every 30 minfor 2 hrs. As shown in FIG. 4B, the expected nalidixic acid mediatedupregulation of genes in the SOS regulon was detected as increasedbioluminescence. In contrast, strains containing fusions to non-DNAdamage responsive genes, osmY and lacZYA, were unaffected by nalidixicacid treatment (FIG. 4A). The strain containing the parental plasmid,pDEW201, without a promoter driving luxCDABE expression produces verylow levels of bioluminescence that are very close to the backgroundmeasured on a ML3000 luminometer for strains grown in 96-wellmicroplates. The apparent basal bioluminescence and upregulation of thisstrain (FIG. 4A) is likely due to cross-talk from an adjacent straincontaining a DNA damage responsive gene fusion.

[0271] High Density Luxarray 0.5

[0272] The clone set of Luxarray 0.5 was used to develop a highlyparallel solid phase assay by printing the clones at high density on theapproximately 8×12 cm membrane. These arrays were used to furtherdevelop the system.

[0273] Printing Density

[0274] In an effort to more closely approximate the final assay system,the clones of Luxarray 0.5 were used to determine the maximum densityone could print the cells at and carry out useful analyses. Arrays wereprinted as above at increasing density and grown for 8 hr at 37° C.Luminescence was imaged using an EagleEye II (Stratagene, La Jolla,Calif.). From visual inspection individual clonal areas of growth couldbe resolved at densities up to 6144 individual spots per membrane. Froma comparison of the size of the growth areas from different densities,it appeared that in the 8 hr growth period, there was a nutrient-limiteffect that resulted in an inversely proportional amount of growth asdensity increases. At this density, there is over two-fold greater thenthe estimated number of transcription units in the entire E. coligenome. Thus a single 8×12 cm array could represent the entire E. coligenome in duplicate with capacity left for controls. Alternatively fullgenome coverage of two different strains or different species could beprinted on the same array.

[0275] High Density Array Image Collection

[0276] The essence of this assay is to collect an image of the signalgenerated from reporter constructs such that the signal intensity can besubsequently quantified. This requires not only that the collectionparameters (focal plane, magnification, integration time, and algorithm)are constant but also that the downstream image analysis software hasthe ability to process the images generated. The most common applicationof this assay, chemical perturbation, requires physically relocating amembrane from one culture plate to another. This results in images withminimal X-Y positional registration. Several commercially availableproducts can efficiently process these images. ArrayVision™ (ImagingResearch, Toronto, Canada) and ImageQuant (Molecular Dynamics,Sunnyvale, Calif.) are two examples of appropriate software packages.

[0277] High Density Luxarray 0.5 Nalidixic Acid Perturbation

[0278] The clones of Luxarray 0.5 were used to print arrays as describedabove at a 4×4 density. That is to say that each single spot as in FIG.3 was replicated 16× in a 4×4 subarray. This resulted in each clonebeing printed 128 times on different areas of the array. Arrays wereprinted in triplicate from fresh overnight cultures onto membranes onplates containing LB media. After 6 hr of growth at 37° C. membraneswere moved to prewarmed plates containing either LB media or LB mediasupplemented with 5 μg/ml nalidixic acid (NA) previously demonstrated tocause detectable induction of responsive promoters for a wide range ofpromoter strength (FIG. 4). Cultures were replaced at 37° C. to continuegrowing. Images were collected for each array every two hours from 0-8hr after relocation using a cooled CCD camera (FluorChem 8000: f.0.85lens, 2 min exposure. AlphaInnotech). Spot intensity was determinedusing ArrayVision™ (Imaging Research, Toronto, Canada). FIG. 5 shows theresults for selected strains containing reporter gene fusions.

[0279] As found with the low density experiments quantified with theluminometer, the expected responses for each clone were welldemonstrated. The five documented DNA damage-responsive reporterconstructs clearly show an upregulation of expression. In contrast,light production from the strain carrying the lac promoter fusion aswell as several strains carrying other promoter fusions was decreased.This decreased bioluminescence likely reflected the decreased growth andmetabolism of the nalidixic acid treated strains and demonstrates thespecificity of the upregulation of the SOS-regulon gene fusions.Furthermore, the signal from the strain containing the parental plasmidis of dramatically lower magnitude than that of strains withpromoter-lux fusions, thus, demonstrating the advantage of the cooledCCD camera for data capture.

[0280] Lux Array 1.0, a Highly Parallel Promoter Activity Assay.

[0281] Selection of a Maximal Non-Redundant Set of Lux Gene Fusions.

[0282] The genome-registered Lux-A luxCDABE gene fusion collection(described in Example 1) provided a list of 4988 plasmid-borne genefusions, each with boundary information relative to the E. coli genomeand the orientation relative to the lux operon. An operably linked or afunctional construct was defined as one consisting of a genomic fragmentencompassing a promoter adjacent to the promoterless lux operon in anorientation that causes transcription initiated at the promoter toproceed into and through the lux operon. Therefore, to identify thefunctional subset of the collection, criteria were computationallyapplied to filter the list of clones first for functionality and secondfor redundancy. A list of definitions of documented and predictedoperons (Thieffry et al. (1998) Bioinformatics 14:391-400) was used todefine genomic coordinates of the operons as the translational startcodon position of the first open reading frame (ORF) in the operon andthe translational stop codon position of the last ORF in the operon.Additional information included the strand on which the operon is coded(direction of transcription), and gene names (common or “b” number). Thelux gene fusions were filtered computationally using the followingassumptions; (i) a functional transcriptional fusion would result fromany genomic fragment starting greater than 50 base pairs upstream of thestart codon of the first ORF and ending anywhere between the start codonof the first ORF in the operon and the stop codon of the last ORF in theoperon, thereby eliminating the occurrence of a transcriptional stopsignal in the construct between the promoter and the lux operon; and(ii) the promoter contained in the genomic fragment must face in thecorrect orientation relative to the lux operon (pictorially representedin FIG. 6). Finally, in cases when more then one clone fit the criteriafor a single operon, only the one construct containing the genomicfragment representing the greatest amount of upstream sequence wasretained thereby eliminating redundancy. The PERL code used and theresulting list of 689 selected gene fusions are found in Scheme 1 andTable 18 (following Example 7). These fusions represent 27% of the 2584known and predicted transcriptional units in the E. coli genome.Individual cultures of strains containing the identified gene fusionswere rearrayed from the ninety original culture plates to create a setof sixteen 96-well microplates containing all the identified fusions,duplicated with side by side symmetry, including appropriately placedcontrols. These 16 plates represent were used to generate the cellulararrays for use in subsequent analyses.

[0283] Preparation of Reporter Array.

[0284] The E. coli strains were grown overnight at 37° C. in 40 μl LBmedium supplemented with 100 μg/ml ampicillin in a set of sixteen96-well dishes. These cultures were designated “printing plates” andwere used as the cell source to manufacture the arrays. Porous membranes(Biodyne B, Nunc) were sterilized with UV illumination for 10min thenplaced in contact with pre-warmed of solid LB growth media in a culturedish (OmniTray, Nunc). Printing of 4×4 subarrays was accomplished usinga BioMek 2000 (Beckman Coulter) equipped with a High Density ReplicationTool (HDRT). Sterilization in between transfers was accomplished bysoaking the pins successively in 0.2% SDS in water, sterile water, and70% ethanol. After sterilization, the pins are air-dried prior to thenext transfer. The E. coli strains in the LuxArray were printed onto anapproximately 8×12 cm membrane in two sets of triplicates.

[0285] Growth in the Array.

[0286] The growth rate of individual colonies was evaluated becauseinitial experiments clearly demonstrated a large distribution of growthrates for the reporter strains in the system. In order to differentiatebetween clone-dependent or system-dependent sources of this variability,bioluminescent cellular arrays were generated in triplicate on differentdays using LB agar media containing 10 μg/ml tetrazolium blue. Theproduct generated when live cells reduce tetrazolium blue is aninsoluble blue precipitate. This greatly increased contrast between thecells and the media simplifying direct imaging of the cells by normallight. For each of the triplicate experiments, each clone was visuallyscored to determine the size of the growth generated during 8 hrs ofincubation at 37 C (data not shown). Variability was clearlyclone-specific and very consistent from day to day. As the majority ofproposed analyses are relative measurements, and inter-clone comparisonsare unlikely, this type of growth variability does not effect theapplicability or robustness of the overall assay system. No furtherattempts were made to determine the source of the variability, howeverit can be assumed that it is a result of the plasmid constructs carriedby the clones.

[0287] Bioluminescence of LuxArray 1.0.

[0288] Images of the bioluminescence were using a cooled CCD camera(FluorChem 8000: f.0.85 lens, 2 min exposure, AlphaInnotech) withoutadditional light source. The bioluminescence from one such array grownfor 16 hours on rich media is shown in FIG. 7. The array in FIG. 7 showsside by side replicate subarrays that include control strains, a strainwith lacZYA promoter fusion and a strain containing the parental plasmid(pDEW201).

[0289] Identification of Nalidixic Acid Responsive Gene Fusions UsingLuxArray 1.0.

[0290] The antibiotic nalidixic acid, an inhibitor of DNA gyrase knownto be a effective inducer of the SOS DNA damage stress response was usedto demonstrate the utility of this array. Arrays were printed from freshovernight cultures onto membranes on plates containing LB media. After 6hr of growth at 37° C. membranes were moved to pre-warmed platescontaining either LB media or LB media supplemented with 5 μg/mlnalidixic acid then replaced at 37° C. to continue growing. Images werecollected for each array every two hours from 0-8 hr after relocationusing a cooled CCD camera (FluorChem 8000: f.0.85 lens, 2 min exposure,AlphaInnotech).

[0291] Spot intensity of each image was determined using ArrayVision™(Imaging Research, Toronto, Canada) and the resultant pixel densitymeasurements imported into a template with identifiers for each spot.The average signal for each of the triplicate spots was calculated. Thebackground signal, which results from cross illumination of neighboringspots, was calculated by finding the median of the 24 spots containing astrain with the parental plasmid on each of the triplicate arrays andthe calculating the average of the three medians. This background signalwas calculated at each of the time points and subtracted from eachmeasurement at the corresponding time point. All negative numbers wereconverted to zero.

[0292] Data normalization to account for inhibition of growth bynalidixic acid was accomplished by finding the sum of the averagedsignals of each spot in the array for each treatment at each time point.A normalization factor (NF) was calculated as follows:

NF=Total array signal (time zero, LB control)/Total array signal (timex, condition y)

[0293] Each measurement was multiplied by NF to yield a normalizedsignal. Ratios the nalidixic acid treated spot to the correspondingcontrol spot were calculated using the normalized data.

[0294] The ratios of the normalized data were compared for each of theduplicate spots resulting from independent cultures in the array.Putative nalidixic acid upregulated gene fusions were selected as thosefor which the ratio in both duplicate spots was at least 2 at both the 2hour and 4 hour time points. Twelve gene fusions were selected by thesecriteria (Table 15).

[0295] These twelve gene fusions include three well-characterizedmembers of the SOS regulon as well as several fusions to promoters notpreviously known to be upregulated by nalidixic acid treatment. The DNAsequence of plasmid DNA isolated from each of these twelve culturesreconfirmed the identity of each inserted DNA. The LuxArray contains atotal of six gene fusions to SOS regulon operons, all of which would beexpected to be upregulated by nalidixic acid. Three were, thus, scoredfalsely as negatives. An examination of the data showed that two ofthese false negatives were reproducibly upregulated but not at level ofthe selection criteria; a fusion to uvrD was upregulated by 1.6 and 1.9fold at 4 hours of nalidixic acid treatment, while one to ruvA wasupregulated 1.7 and 2.1 fold at that time point. The third did not haveconsistent responses; the ratio of nalidixic acid treated to untreatedfor the dinF-lux fusion was found to be 4.2 in one of duplicate spotsand 0.7 in the other.

[0296] Validation of Nalidixic Acid Upregulated Gene Fusions byRetesting in Liquid Medium.

[0297] Each of the newly identified putative nalidixic acid upregulatedgene fusions and two of the known SOS gene fusions were retested usingexponentially growing cultures in liquid medium with sevenconcentrations of nalidixic acid (80 μg/ml in two fold dilutions to 1.2μg/ml). Several concentrations of nalidixic acid were used because thedifferences in responses between liquid and solid growth were not known.Light production of 100 μl duplicate cultures at 37° C. was quantitatedusing a 96 well plate luminometer (Luminoskan Ascent, Labsystems). Table15 shows the results expressed as ratios of the signal from thenalidixic acid treated cultures to the untreated control at 2 hours atthe concentration that yielded the maximal response. Also shown is thenumber of concentrations of nalidixic acid tested that resulted inresponse ratios of 1.5 or greater. It should be noted that the liquidmedium tests were not corrected for growth inhibition by nalidixic acid.Using a standard of a maximal response ratio of at least 1.8 andresponses ratios that were >1.5 fold at 3 or more concentrations, 7 ofthe 9 putative novel nalidixic acid upregulated gene fusions were shownto be reproduced in liquid medium.

[0298] Mitomycin C Responses and Effect of lexAind Mutation.

[0299] Mitomycin C, a DNA damaging compound with a different mechanismof action from nalidixic acid, was used to determine if these newlydiscovered nalidixic acid upregulated gene fusions were generallyresponsive to DNA damage. In addition, the effect of a lexAind mutationwas tested to determine if any of these were part of the SOS regulon.The expectation is that SOS regulon member will be induced by bothnalidixic acid and mitomycin C in a manner that is dependent on lexAfunction (Walker, G. C., (1996) Escherichia coli and Salmonella:Cellular and Molecular Biology ASM Press). As shown in FIG. 9, a fusionof the promoter region of b1728 to luxCDABE was clearly induced by bothnalidixic acid and mitomycin C in the lexA+ host, but was not induced bythese chemicals in the lexAind host strain. Thus, these resultsdemonstrate that upregulation by nalidixic acid as well as mitomycin Cis controlled by LexA. This is consistent with the observation ofupregulation of the b1728 mRNA transcript upon mitomycin C treatment ina LexA dependent fashion (Fernández de Henestrosa et al. (2000). Mol.Microbiol. 35:1560-1572).—Likewise, two gene fusions, those to oraA andyigN were identified as new members of the SOS regulon (Table 16).Interestingly, four of the nalidixic acid responsive gene fusions werenot upregulated by mitomycin C in the lexA+ host suggesting that theyare not generally DNA damage responsive, but rather are morespecifically responsive to nalidixic acid. Negative, non-DNA damageresponsive gene fusions were also included (Table 16).

[0300] Thus, the LuxArray assay was useful to identify novel nalidixicacid upregulated genes in E. coli. Likewise, this robust assay can beused generally in a fashion parallel to hybridization assays to monitortranscriptional changes. The multiple whole genome scale capacity andrelative simplicity of manufacture allow for significant throughput.This assay is an important addition to efforts making functionalassignment of promoter activity. TABLE 15 Nalidixic acid responses onsolid and in liquid medium Solid Solid Nal conc Liquid Solid expt exptexpt Solid expt of max expt. # of conc Fusion to gene or NA/LB NA/LB,NA/LB, NA/LB, induction NA/LB with > 1.5 operon: Lux ID t = 2 hr t = 4hr t = 6 hr t = 8 hr in liquid t = 2 hr fold up Known SOS regulonmembers: dinG ybiB lux-a.pk0024.f5 3.4 4.2 3.1 2.6   5 ug/ml 4.18 6lux-a.pk0024.f5 2.5 3.7 3.5 2.9 dinP lux-a.pk055.a3 3.1 5.1 4.2 3.1   5ug/ml 7.60 7 lux-a.pk055.a3 5.5 11.0 8.1 6.3 uvrA lux-a.pk0001.b6 2.52.3 1.6 1.0 nd nd nd lux-a.pk0001.b6 2.9 2.4 1.1 0.6 Nalidixic acidupregulated on both the solid LuxArray and in liquid culture b1169lux-a.pk0015.d6 2.3 6.4 2.7 0.6  20 ug/ml 2.71 6 lux-a.pk0015.d6 2.2 4.11.9 0.8 b1728 lux-a.pk033.c5 3.7 4.3 3.2 2.6   5 ug/ml 3.67 7lux-a.pk033.c5 2.1 3.1 3.3 2.5 b1936 lux-a.pk0019.gl 2.5 7.7 9.7 7.1  10ug/ml 2.34 6 lux-a.pk0019.gl 4.4 8.2 15.5 10.2 lpxA lpxB mhB dnaElux-a.pk061.c3 3.5 3.1 2.8 1.7 2.5 ug/ml 1.82 3 lux-a.pk061.c3 2.7 3.53.0 1.9 oraA lux-a.pk058.f5 5.1 7.9 5.9 3.9  10 ug/ml 7.19 7lux-a.pk058.f5 4.7 7.7 5.6 3.8 yaaF lux-a.pk031.e7 2.1 2.0 1.3 1.0 2.5ug/ml 1.85 3 lux-a.pk031.e7 2.0 2.1 1.8 1.5 yigN lux-a.pk046.f11 2.1 2.50.3 0.1 1.2 ug/ml 2.54 5 lux-a.pk046.f11 7.1 4.3 2.5 2.2 Nalidixic acidupregulation not reproduced in liquid culture frvR frvX frvB frvAlux-a.pk046.e6 2.8 2.3 0.8 0.5  20 ug/ml 1.46 0 lux-a.pk046.e6 2.1 2.32.0 1.7 yfhJ fdx hscA yfhE lux-a.pk0019.g2 2.1 2.0 1.7 1.4 1.2 ug/ml1.02 0 lux-a.pk0019.g2 2.1 2.2 1.5 1.3

[0301] TABLE 16 Mitomycin C and Nalidixic acid Responses in lexA+ andlexAind hosts NA MitC Fusion to Ratio, Ratio, gene or Host 2 hr 2 hroperon: Lux ID Strain 10 ug/ml 250 ng/ml Known SOS regulon member: dinGybiB lux- lexA⁺ 4.03 5.01 a.pk0024.f5 lexA^(ind) 0.97 1.15 Nalidixicacid upregulated on both the solid LuxArray and in liquid culture NewSOS regulon members b1728 lux-a.pk033.c5 lexA⁺ 5.17 8.97 lux-a.pk033.c5lexA^(ind) 0.40 0.90 oraA lux-a.pk058.f5 lexA⁺ 11.06 15.27lux-a.pk058.f5 lexA^(ind) 1.06 1.21 yigN lux- lexA⁺ 5.74 9.84a.pk046.f11 lux- lexA^(ind) 0:59 0.93 a.pk046.f11 Nalidixic acidupregulated, not generally DNA damage inducible b1169 lux- lexA⁺ 3.221.12 a.pk0015.d6 lux- lexA^(ind) 2.29 1.73 a.pk0015.d6 b1936 lux- lexA⁺3.25 1.22 a.pk0019.g1 lux- lexA^(ind) 1.55 2.46 a.pk0019.g1 lpxA lpxBlux-a.pk061.c3 lexA⁺ 2.87 1.06 rnhB dnaE lux-a.pk061.c3 lexA^(ind) 2.391.29 yaaF lux-a.pk031.e7 lexA⁺ 1.53 1.02 lux-a.pk031.e7 lexA^(ind) 1.190.99 Negative controls yciG lux-a.pk006.b4 lexA⁺ 0.32 0.56 lexA^(ind)0.67 0.61 lacZ 65.d4 lexA⁺ 0.81 0.80 lexA^(ind) 1.05 1.24

Example 6 Additions to Collections and Alternative Methods of GeneratingCollections of Bacterial Genomic Fragments Fused to a Reporter

[0302] There are several methods for making fusions of bacterialpromoter regions to reporter genes to add to a genome-registeredcollection of randomly generated gene fusions. Some of these methods arealso alternative methods for building a large collection of genefusions.

[0303] Firstly, DNA sequence data from more random gene fusions willresult in identification of additional useful members to a collectionthat is not completely saturated. These additional sequences can begenerated from members of the same originally sequenced library ofgenetic fusions, LuxA for instance, or an independently generatedlibrary. The steps outlined in previous examples allow the genomeregistration of the newly sequenced fusions.

[0304] DNA sequencing of randomly generated fusions will lead toidentification of fusions that would be useful except that theorientation of the chromosomal DNA is inverted, such that the promoterregions of interest are not operably linked to the reporter genes.Selecting such fusions and inverting the orientation of the insert DNAcan significantly enhance the utility of a sequenced collection byadding many more operable linked fusion to the collections. A simple wayto do this is to digest the plasmid DNA with a restriction enzyme thatcuts just outside the cloned region and religate the pieces. Although amixture of plasmids results from this procedure, in many cases thecorrectly oriented plasmid can be found because cells containing it, butnot other possible products, will produce light. This method has theadvantage of avoiding use of the polymerase chain reaction and thusavoiding possible changes in the DNA sequence from amplification.

[0305] This approach was demonstrated by identifying a non-operablylinked clpB fusion in the Lux-A Collection, inverting the chromosomalDNA segment relative to the vector, and comparing an E. coli straincarrying the resultant plasmid to one in the collection with a properorientation of the clpB promoter region. One isolate from the Lux-ACollection, lux-a.pk043.d3 contains the region of the E. coli chromosomewith the clpB promoter, but is oriented in the opposite direction thanrequired to operably link the promoter and reporter genes. Very lowlevels of light production of this strain were measured. In contrast,the strain, lux-a.pk054.c3, that carries the plasmid with the clpBpromoter region in the proper orientation to operably link it to theluxCDABE reporters gene had a high level of light production. PlasmidDNA was isolated from lux-a.pk043.d3, digested with restriction enzymeXmaI, and ligated with T4 DNA ligase. The ligation reaction was used totransform E. coli strain DPD1675 by electroporation. Transformants wereselected by ampicillin resistance. Three percent of the resultanttransformed colonies produced bioluminescence as detected by exposure ofX-ray film. The response to ethanol treatment of two of these lightproducing transformants that putatively contain inverted chromosomalinserts, of lux-a.pk054.c3 (DPD2243) that contains a plasmid with theclpB promoter operably linked to the luxCDABE reporter and of theoriginal lux-a.pk043.d3 were compared. Actively growing cultures at 37°C. in LB medium were treated with 4% ethanol and light production wasquantitated at 37° C. using a microplate luminometer (Dynex ML3000).Table 17 summarizes the average bioluminescence of duplicate samples at38 min after treatment. TABLE 17 Light production by strains containinga clpB-luxCDABE gene fusion resulting from inversion of the chromosomalinsert, and controls Light Light production Culture production in LB +4% Response Strain turbidity¹ in LB² ethanol² Ratio lux-a.pk043.d3 110.0015 0.0025 N/A Inverted 14 1.25 8.17 6.5 lux-a.pk043.d3 Inverted 185.34 29.63 5.5 lux-a.pk043.d3 lux-a.pk054.c3 12 1.37 13.00 9.4 (DPD2243)

[0306] Thus, the presence of a promoter that drives transcription of theluxCDABE operon in the inverted isolates was shown by the lightproduction in LB medium. Furthermore, the expected biological responseof induction of up-regulation by ethanol treatment for this member ofthe heat shock regulon was also demonstrated.

[0307] This inverting method was implemented in a parallel manner to addgene fusions to the Lux-A Collection. First, a list of about 400 genefusions from the Lux-A Collection that contained the correct genomicfragment but in the wrong orientation relative to the lux operon, andwere not present in the current collection was derived. Each of thestrains containing these fusions was picked out of the originalcollection of 8000 fusions and regrown. The bioluminescence of each wasquantitated because, in some cases, the presence of a divergent promoterin the cloned DNA fragment resulted in light production even though theDNA was inverted with respect to the promoter of interest. About half ofthe selected strains produced >0.1 RLU of light following 3 hours ofincubations at 37° C. after inoculation of 100 μl LB medium with 10 μlof culture from a working plate. This suggested that the set of strainswith light production of <0.1 RLU would be easier to pursue initially.Thus, following isolation of plasmid DNA in a 96 well format, XmaIdigestion, and religation, the ligation reaction mixes originating fromstrains with light production of less than 0.1 RLU were sorted intoseparate plates from those that originated from strains with lightproduction greater than or equal to 0.1 RLU. E. coli DPD1675 was madecompetent by calcium chloride treatment and transformed with thereligated plasmid DNA selecting for ampicillin resistance. Placing thepetri plate with the transformant colonies on X-ray film and developingthe exposed film at various time points of contact identified lightproducing transformants. The light producing colonies were purified byisolating single colonies in solidified LB medium containing ampicillinand light production was verified by luminometry. This process wascompleted for 36 plasmids isolated from strains with light production ofless than 0.1 RLU. Plasmid DNA from each of the strains containingputative inverted chromosomal segments was isolated and DNA sequence wasdetermined to assess if the inverted product had been obtained. Thirteenof the 36 plasmids yielded DNA sequence information that had a highscoring hit when compared with the E. coli genomic DNA sequence usingBLASTN (default setting). Of these, five had DNA in the invertedorientation from the original clone.

[0308] There are several potential reasons for this relatively lowsuccess rate. For example, some promoters that are annotated may not befunctional in the conditions used or may not be biologically relevantpromoters. Thus, selection by light production of the inverted clonewould not be successful. In contrast, divergent promoters that were notactive in the liquid culture test for bioluminescence used for sortingmay have been active on the solidified medium used to screen for lightproducing clones. This may have lead to selection of a colony containingthe initial plasmid. Furthermore, there may have been poor plasmid DNAyield, poor cutting, and/or poor religations when done in parallel inmicroplates. This may have lead to DNA concentrations in the ligationssuch that intermolecular ligations were minimized or such that a lownumber of transformant colonies resulted and rare transformants with theinverted insert DNA were not represented. Thus, this approach ofrestriction digestion, religation, transformation and selection of lightproducing transformants has been demonstrated to work. However, furtheroptimization for efficient parallel implementation is required.

[0309] To test inverting of a chromosomal segment in a plasmidcontaining divergent promoters, the backward fusion to yebF was chosen.Obtaining an operably linked reporter gene fusion to yebF was ofparticular interest because the yebF gene is an otherwise unknown openreading frame that was found in a DNA microarray experiment to be highlyinduced by mitomycin C, a DNA damaging agent. In this case, a divergentpromoter to the purT gene was present in the chromosomal fragment andstrains containing the backward yebF fusion produced light. FollowingXmaI digestion, religation and transformation, 10% of the transformantsproduced light. Of these, 20% (or 2% of the initial transformants) werefound to be highly induced by nalidixic acid, another DNA damagingagent, This result strongly suggesting that the desired inversion of thechromosomal segment had occurred. This was verified by DNA sequenceanalysis. Thus, inverting chromosomal segments containing divergentpromoters is possible and is facilitated if the two promoters can bedistinguished by their biological activity.

[0310] An alternative inverting procedure uses plasmid DNA from clonesrequiring reorientation as templates for the polymerase chain reaction(PCR). Universal PCR primers were designed which hybridize specificallywith the pDEW201 plasmid flanking the genomic fragment cloning site (BamHI). Use of the first primer, pDEWE2S (SEQ ID NO:3,5′-GGAATTGGGGATCGGAGCTCCCGGG-3′), an EcoRI site (GAATTC) is converted toa SacI site (GAGCTC) via an internal AT to GC mismatch. Use of thesecond primer, pDEWS2E (SEQ ID NO:4, 5′-GAATGGCGCGAATTCGGTACCCGGG-3′),results in the conversion of the SacI site to an EcoRI site via aninternal GC to AT conversion. Thus the resultant PCR products from anypDEW201 clone can be digested with EcoRI and SacI and ligated intoEcoRI- and SacI-digested pDEW201. The resultant plasmid contains theoriginal chromosomal segment in the opposite orientation of the originalclone, relative to the lux operon. As the primers are specific to thevector they can be used for all clones and are amenable to highthroughput (96-well plate) approaches.

[0311] Construction, Sequencing and Registering an Additional RandomLibrary of E. coli Genomic Fragments Fused to a luxCDABE Reporter.

[0312] An independent random library of E. coli genomic fragments inplasmid pDEW201 was constructed and called the LuxZ library. ChromosomalDNA isolated from E. coli strain W3110 (Ernsting et al., (1992) J.Bacteriol. 174:1109-1118) was partially digested with the restrictionenzyme Sau3A1, size fractionated by agarose gel electrophoreses, and afraction with an average size of approximately 0.7 KB isolated. Thisfraction was ligated to pDEW201 that had previously been digested withBamHI and treated with calf intestinal alkaline phosphatase. Theligation products were used to transform ultracompetent E. coli XL2Bluecells (Stratagene) to ampicillin resistance using the protocol providedby Stratagene. Preliminary characterization of individual XL2Bluetransformants that were picked in random indicated that a largepercentage (16 of 16) contained insert DNA with sizes ranging from 0.1to 1.5 KB. Approximately 150,000 of these transformants were pooled andused as a source of heterogeneous LuxZ library plasmid DNA isolatedusing Qiagen tip20 columns (Qiagen Corp). This plasmid DNA pool wasdiluted 100-fold then used to transform E. coli DPD1675 (Van Dyk andLaRossa (1998) Methods in Molecular Biology: Bioluminescence Methods andProtocols, Humana Press inc. vol. 102:85-95) by electroporation.Electrocompetent DPD1675 cells were prepared starting with 1 liter of LBculture at approximately 2.4×10⁸ cells/ml. Following chilling on ice,the cells from this culture were collected by centrifugation at anaverage relative centrifugal force of 6,555 for 5 minutes. The cellswere washed twice ice-cold, sterile distilled water (1 liter for thefirst wash and 500 ml for the second wash) and centrifuged. Then thecells were washed with 10 ml of ice-cold, sterile 10% glycerol indistilled water and centrifuged. The final resuspension of the cellpellet was done with ice-cold, sterile 10% glycerol in distilled waterto yield a final volume of 2.1 ml. 55 μl aliquots were prepared, quickfrozen in dry ice and ethanol, then stored at −80° C. until used. Onesuch aliquot was thawed on ice, 1.0 μl of the diluted LuxZ libraryplasmid DNA was added, and the cells and plasmid DNA were transferred toan electroporation cuvette (Biorad, Gene Pulser®/E. coli Pulser™) onice. The capped cuvette was electroporated with 1.85 volts at acapacitance of 25 μF then 0.5 ml of SOC medium (per liter: 20 gtryptone, 5 g yeast extract, 0.5 g NaCl, 2.5 mM KCl, 2.5 mM MgCl₂, 20 mMglucose, pH 7.0) was added. A 30 minute phenotypic expression time at37° C. was used to minimize the presence of siblings. Following thisincubation, 500 μl of 24% sterile glycerol in water was added and 100 μlaliquots were prepared, frozen on dry ice, and stored at −80° C. untilused. These aliquots were thawed, plated on LB medium containingampicillin, and 4608 single colonies were picked for use as inoculum forcultures grown in 80 μl 1× freezing medium (LB with 100 μg/mlampicillin, 5.54% glycerol, 36 mM K₂HPO₄, 13.2 mM KH₂PO₄, 1.7 mMNaC₆H₅O₇:2H₂O, 40 mM MgSO₄, 6.8 mM NH₄SO₄) in the wells of 384 wellmicroplates. These plates were stored at −80° C. until used forinoculation and growth to saturation in Terrific Broth (Gibco-BRL, Inc)containing 100 μg/ml ampicillin in 96 well deep-well plates. Plasmid DNAwas extracted from the cultures using the Qiagen R.E.A.L.™ prep methodwith the following modification: after lysis of cells, the plates wereplaced in a boiling water bath for 5 minutes and then rapidly chilled inan ice-water bath before precipitation with Buffer R3. This modificationprevented degradation of the plasmid DNA by the nucleases present in thenon-endA host strain, DPD1675. DNA sequencing reactions were performedwith approximately 1 μg of plasmid DNA under standard ABI Prism™dRhodamine DyeTerminator Ready Reaction conditions with the primerspDEW201.forward (SEQ ID NO:1, 5′-GGATCGGAATTCCCGGGGAT-3′) andpDEW201.reverse (SEQ ID NO:2, 5′-CTGGCCGTTAATAATGAATG-3′) to obtainsequence information from each end of the insert. DNA sequences weredetermined on ABI377™-XL 96-lane upgraded Sequencers under 4× runconditions on 5% PAG (polyacrylamide gel) LongRanger™ (FMC, Inc.) gelsand analyzed with ABI software. DNA sequences were transferred to a UNIXbased utility for further analysis. The homology search for the sequencefrom the beginning and end of each Lux-Z clone (in both orientations)and registration to the E. coli genome was done as described in Example1 resulting in 1799 additional genome-registered gene fusions.

Example 7 Lux Array 1.04, a Genome-Wide, Promoter Activities Assay inLiquid Medium, and its Use to Discover a Limonene Sensor

[0313] Selection of Additional, Non-Redundant Lux Gene Fusions to ExpandLuxArray 1.0.

[0314] Operably linked gene fusions were selected from thegenome-registered LuxZ gene fusion collection described in Example 6using the computational filter described in Example 5. This list wasthen compared with the current list of promoters contained in Lux Array1.0 to eliminate redundancy. This process resulted in identification of149 gene fusions useful to expand the LuxArray.

[0315] In addition, the five gene fusions generated by inversion ofpreviously sequenced gene fusions as described in Example 6 and six genefusions from other sources were added. Table 19 lists these additional160 gene fusions that together with the gene fusions in LuxArray 1.0yielded LuxArray 1.04. The 849 gene fusions in Lux Array 1.04 thusrepresent 33% of the 2584 known and predicted transcriptional units inthe E. coli genome. Individual cultures of strains containing the newlyidentified gene fusions were added to empty wells of the existingmicroplate number 15 and new microplates numbered 17 to 19 were madewith duplicated side by side symmetry. The former microplate 16 waseliminated because it contained only strains with the parental plasmidand sterile controls. Thus, the Lux Array 1.04 is contained in 18microplates representing the cellular array. These were stored at −80°C. in freezing medium.

[0316] Limonene Stress of the Reporter Array in Liquid Culture.

[0317] Microplates from −80° C. containing the LuxArray 1.04 E. colistrains were thawed and used to inoculate 100 μl LB medium supplementedwith 100 μg/ml ampicillin in Costar#3595 flat bottomed 96-wellmicroplates. Following overnight growth at 37° C., the plates werevisually examined and any wells with little or no turbidity wererecorded. Then these cultures were diluted by transfer of 15 μl to 135μl LB medium in Costar #3595, flat bottom 96-well microplates and weremoderately swirl shaken on an IKA Schuttler MTS 4 shaker at setting 400for two hrs at 37° C. in a humidified box. Twenty μl of each theseactively growing cultures were transferred to the corresponding well insets of three microplates, one containing 80 μl of LB medium, anothercontaining 80 μl of LB medium saturated with limonene at roomtemperature, and the third containing 40 μl of LB medium saturated withlimonene and 40 μl of LB medium. An initial (zero time) reading wastaken using an MLX microplate luminometer (Dynex) pre-warmed to 37° C.Then the plates were placed at 37° C. without shaking. Additionalbioluminescent measurements were taken at 45, 90, and 135 minutes. Tolimit the number of microplates handled each day, LuxArray 1.04microplates 1 to 5, microplates 6 to 10, microplates 11 to 15, andmicroplates 17 to 19 were used on each of four different days.

[0318] Data Analysis of Limonene Induced Responses.

[0319] The data were normalized to correct for growth inhibition causedby limonene on the basis of each individual day that data was collected.The bioluminescence (RLU) of each individual culture for each day ofexperimentation under each of the three conditions, were added todetermine the daily array total RLU. These were used to find a dailynormalization factor (DNF) as follows:

DNF=Daily total array signal (time zero, LB control)/Daily total arraysignal (time x, condition y)

[0320] Each measurement from the corresponding day, time point, andcondition was multiplied by DNF to yield a normalized signal. The datafrom wells that were scored to have little or no growth were not used infurther analysis. The normalized data were analyzed with GeneSpring(Silicon Genetics) software, which averaged the signal from each of theduplicate cultures for each reporter gene fusion.

[0321] Overall, there was little to no upregulation of gene expressionunder the condition of 40% saturated limonene in LB medium. Thus,patterns of gene expression from the 80% saturated limonene in LB mediumwere further examined. Lists were made of gene fusions that wereupregulated by 2 fold or more at each time point. These lists wereexamined and gene fusions with very low normalized RLU values (<0.012RLU) at all conditions were eliminated. The upregulated gene fusions arelisted in Table 20, which uses the name of the gene in each operon withthe smallest b# to identify each gene fusion. The designation of “>2×”is given for upregulation while “*” is listed if the expression was lessthan 2 fold increased. At 45 minutes, the expression of fourteen genefusions was increased; none of these was increased 3 fold or higher. At90 minutes, 37 gene fusions were induced; these included all but one ofthe gene fusions observed at 45 minutes. However, the most highlyupregulated gene fusions at 90 minutes were not previously observed tobe upregulated at 45 minutes. These were fusions of the luxCDABEreporter to the promoter regions of uhpT (3.8×), nirB (3.7×) and narK(3.6×). At 135 minutes, 39 gene fusions were induced, which included 13fusions not observed at 45 or 90 minutes. At this time point, the mosthighly upregulated gene fusions were those to the promoter regions ofuhpT (7.7×), nirB (4.1×) and katG (4.1×). Thus, the gene fusion to thepromoter region of uhpT was the most highly upregulated at both the 90and 135 minute time points.

[0322] Verification of uhpT-Lux Upregulation by Limonene.

[0323]E. coli strain DPD3228 is identical to lux-a.pk034.b9 the straincontaining the uhpT-luxCDABE gene fusion. This strain was grownovernight at 37° C. in LB medium containing 100 μg/ml ampicillin anddiluted the following day into LB medium and grown to log phase at 37°C. 20 μl aliquots of this culture were added to 80 μl of LB mediumsaturated with limonene, to 80 μl of a series of two fold dilutions intoLB medium of LB saturated with limonene, and to 80 μl of LB medium.Light production was measured in a Luminoskan Ascent (Labsystems)microplate luminometer at 37° C. FIG. 10 shows the result. Similar tothe initial LuxArray observations, a late response to the presence oflimonene was observed. This response was maximal at the highestconcentration of limonene tested, but was also detected when limonenewas present at 40% or 20% of saturating amounts in LB medium. Thus, theupregulation of bioluminescence from this strain by limonene wasconfirmed, demonstrating its utility as a sensor for limonene.

[0324] Scheme 1: PERL Script Used to Filter the Lux Clone Collection forFunctional Reporter Constructs #Filename:luxfilter2 #This perl script isdesigned to compare an operon list #containing genome coordinates anddirectionality #and clone ID to a list of clones containing the same andoutput #their intersect to a new file open (OPERONS, “< operons_gg.txt”)∥ die “can't open operon list: $!”; open (OUTPUT, “> full_list2.txt”) ∥die “can't open output file: $!”; open (CLONES, “< luxclones.txt”) ∥ die“can't open clone list: $!”; @clones = <CLONES>; close CLONES; while($line = <OPERONS>){   print OUTPUT $line;   print $line;   if ($line =˜/complement/) {     ($start) = $line =˜ /\d+\.\. (\d+)/;     ($op_end) =$line =˜ /(\d+)\.\.\d+/;     $promoter = $start+50;     foreach $clone(@clones) {       if ($clone =˜ /\t-\t/) {         ($clone_start) =$clone =˜   /\d+\t(\d+)\t\d+/;         ($clone_end) = $clone =˜          /(\d+)\t\d+\t\d+/;         if (($clone_start > $promoter) &&10    ($clone_end > $op_end) &&           ($clone_end < $start)) {          print OUTPUT $clone;           print $clone;         }       }    }   } else {     ($start) = $line =˜ /(\d+)\.\.\d+/;     ($op_end) =$line =˜ /\d+\.\.(\d+)/;     $promoter = $start−50;     foreach $clone(@clones) {       if ($clone =˜ /\+/) {         ($clone_start) = $clone=˜   /(\d+)\t\d+\t\d+/;         ($clone_end) = $clone =˜          /\d+\t(\d+)\t\d+/;         if (($promoter > $clone_start) &&   ($clone_end < $op_end) &&          ($clone_end > $start)) {          print OUTPUT $clone;           print $clone;         }       }    }   } } close OUTPUT; close OPERONS;

[0325] TABLE 18 Luxarray 1.0 Clone Collection Coding Lux ID strandOperon lux-lacZ − complement(360473 . . . 365529) lux-a.pk007.b9 −Operon complement(3399029 . . . 3400969) /note = “predicted operon”/note = “ordered genes contained in the operon: yhdA ” lux-a.pk0017.h6 +Operon (510865 . . . 513092) /note = “predicted operon” /note = “orderedgenes contained in the operon: b0485 b0486 ” lux-a.pk0022.c6 − Operoncomplement(2325387 . . . 2334712) /note = “predicted operon” /note =“ordered genes contained in the operon: b2225 b2226 b2227 b2228 b2229yfaA ” lux-a.pk0026.g3 − Operon complement(120178 . . . 121551) /note =“documented aroP operon” lux-a.pk0032.g2 + Operon (4161218 . . .4171626) /note = “predicted operon” /note = “ordered genes contained inthe operon: btuB murI murB birA ” lux- − Operon complement(1067734 . . .1073234) /note = “predicted a.pk0037.d11 operon” /note = “ordered genescontained in the operon: b1006 b1007 b1008 b1009 b1010 b1011 b1012 ”lux- + Operon (342108 . . . 343157) /note = “predicted operon” /notea.pk0041.f10 = “ordered genes contained in the operon: b0325 ”lux-a.pk047.d10 Operon complement(4592507 . . . 4593313) /note =“predicted operon” /note = “ordered genes contained in the operon: yjjM” lux-a.pk052.f3 + Operon (3246594 . . . 3248016) /note = “predictedoperon” /note = “ordered genes contained in the operon: yqjC yqjD yqjEb3100 ” lux-a.pk058.f5 − Operon complement(2820162 . . . 2820662) /note= “predicted operon” /note = “ordered genes contained in the operon:oraA” lux-a.pk066.a3 − Operon complement(1998496 . . . 2001629) /note =“predicted operon” /note = “ordered genes contained in the operon: fliZfliA fliC ” lux-a.pk072.e2 + Operon (2543793 . . . 2547426) /note =“predicted operon” /note = “ordered genes contained in the operon: b2428b2429 b2430 ” lux-a.pk078.c5 − Operon complement(3117613 . . . 3119295)/note = “predicted operon” /note = “ordered genes contained in theoperon: b2975 ” lux-a.pk086.e2 − Operon complement(4311389 . . .4322743) /note = “documented phnCDE-b4103-phnFGHIJKLMNOPQ operon” lux- −Operon complement(1734145 . . . 1735314) /note = “predicted a.pk0001.a11operon” /note = “ordered genes contained in the operon: b1657 ”lux-a.pk007.c1 − Operon complement(2255449 . . . 2257316) /note =“predicted operon” /note = “ordered genes contained in the operon: yeiNyeiC ” lux-a.pk0018.a4 + Operon (3714927 . . . 3715913) /note =“predicted operon” /note = “ordered genes contained in the operon: yiaE”lux-a.pk0022.d3 + Operon (801110 . . . 802543) /note = “predictedoperon” /note = “ordered genes contained in the operon: b0770 ”lux-a.pk026.e11 + Operon (84191 . . . 87848) /note = “documentedleuO-ilvIH operon” lux-a.pk032.g4 + Operon (214291 . . . 215979) /note =“predicted operon” /note = “ordered genes contained in the operon: yaeQyaeJ cutF ” lux-a.pk037.d4 + Operon (89634 . . . 103153) /note =“predicted operon” /note = “ordered genes contained in the operon: yabByabC ftsL ftsI murE murF mraY murD ftsW murG murC ddlB ”lux-a.pk041.f12 + Operon (3542470 . . . 3543201) /note = “predictedoperon” /note = “ordered genes contained in the operon: yhgH ”lux-a.pk047.d4 − Operon complement(3334196 . . . 3334459) /note =“predicted operon” /note = “ordered genes contained in the operon: yrbA”lux-a.pk052.g10 + Operon (4233811 . . . 4237309) /note = “predictedoperon” /note = “ordered genes contained in the operon: yjbF yjbG yjbH”lux-a.pk058.h10 − Operon complement(578407 . . . 578859) /note =“predicted operon” /note = “ordered genes contained in the operon: b0558” lux-a.pk066.b10 − Operon complement(4297143 . . . 4300516) /note =“predicted operon” /note = “ordered genes contained in the operon:yjcPyjcQ ” lux-a.pk072.e3 + Operon (797809 . . . 7988040 /note =“predicted operon” /note = “ordered genes contained in the operon: ybhE” lux-a.pk078.d2 − Operon complement(1336594 . . . 1337184) /note =“predicted operon” /note = “ordered genes contained in the operon: ribA”lux-a.pk086.h2 − Operon complement(3390094 . . . 3393895) /note =“predicted operon” /note = “ordered genes contained in the operon: yhdPyhdR ” lux-lacZ complement(360473 . . . 365529) lux-a.pk007.d12 − Operoncomplement(2111456 . . . 2112349) /note = “predicted operon” /note =“ordered genes contained in the operon: galF” lux-a.pk0018.a6 + Operon(4140109 . . . 4141523) /note = “predicted operon” /note = “orderedgenes contained in the operon: frwC frwB ” lux- + Operon (2693959 . . .2695377) /note = “predicted operon” a.pk0022.g11 /note = “ordered genescontained in the operon: yfhD ” lux-a.pk027.b11 − Operoncomplement(3598659 . . . 3601874) /note = “documented ftsYEX operon”lux-a.pk032.h3 + Operon (274525 . . . 276871) /note = “predicted operon”/note = “ordered genes contained in the operon: b0260 b0261 ”lux-a.pk037.d6 + Operon (4401964 . . . 4402161) /note = “predictedoperon” /note = “ordered genes contained in the operon: b4176 ”lux-a.pk041.g11 − Operon complement(2904665 . . . 2905963) /note =“predicted operon” /note = “ordered genes contained in the operon: eno”lux-a.pk047.e10 + Operon (4152580 . . . 4155802) /note = “documentedargCBH operon” lux-a.pk052.g3 − Operon complement(3852741 . . . 3853934)/note = “predicted operon” /note = “ordered genes contained in theoperon: yidF yidG yidH ” lux-a.pk059.a8 + Operon (621523 . . . 622773)/note = “predicted operon” /note = “ordered genes contained in theoperon: ybdA ” lux-a.pk066.b12 + Operon (284619 . . . 287623) /note =“predicted operon” /note = “ordered genes contained in the operon: b0270b0271 ” lux-a.pk072.f11 − Operon complement(2061410 . . . 2063786) /note= “documented cobUST operon” lux-a.pk078.d7 − Operon complement(3092119. . . 3093144) /note = “predicted operon” /note = “ordered genescontained in the operon: b2950 ” lux-a.pk087.b5 − Operoncomplement(1952602 . . . 1956156) /note = “predicted operon” /note =“ordered genes contained in the operon: bisZ b1873 ” lux-a.pk0001.a2 −Operon complement(3767870 . . . 3769371) /note = “predicted operon”/note = “ordered genes contained in the operon: yibH yibI ”lux-a.pk007.d2 − Operon complement(2506481 . . . 2507446) /note =“predicted operon” /note = ”ordered genes contained in the operon: glk ”lux-a.pk0018.b1 + Operon (4048927 . . . 4049436) /note = “predictedoperon“ /note = “ordered genes contained in the operon: yihI ”lux-a.pk0022.g7 − Operon complement(783105 . . . 784046) /note =“predicted operon” /note = “ordered genes contained in the operon: b0752” lux-a.pk027.c11 + Operon (1779419 . . . 1782701) /note = “predictedoperon” /note = “ordered genes contained in the operon: b1699 b1700 ydiD” lux-a.pk033.a3 − Operon complement( 1978212 . . . 1980411) /note =“documented otsAB operon” lux-a.pk037.e3 − Operon complement(3290116 . .. 3290976) /note = “predicted operon” /note = “ordered genes containedin the operon: yraL” lux-a.pk041.g5 − Operon complement(1551996 . . .1553720) /note = “predicted operon” /note = “ordered genes contained inthe operon: sfcAft lux-a.pk047.e11 + Operon (2932257 . . . 2938121)/note = “documented fucPIKUR operon” lux-a.pk052.g6 − Operoncomplement(317900 . . . 319252) /note = “predicted operon” /note =“ordered genes contained in the operon: b0304 ” lux-a.pk059.b4 − Operoncomplement(3302214 . . . 3303458) /note = “documented mtr operon”lux-a.pk066.c7 + Operon (87860 . . . 89032) /note = “predicted operon”/note = “ordered genes contained in the operon: fruL fruR ”lux-a.pk072.f2 − Operon complement(3553466 . . . 3555711) /note =“predicted operon” /note = “ordered genes contained in the operon: yhgjyhgK yhgL ” lux-a.pk078.d9 + Operon (4257900 . . . 4258885) /note =“predicted operon” /note = “ordered genes contained in the operon: yjbLyjbM ” lux-a.pk087.b7 + Operon (594823 . . . 596196) /note = “predictedoperon” /note = “ordered genes contained in the operon: b0572 ” lux-lacZcomplement(360473 . . . 365529) lux-a.pk007.d3 + Operon (794312 . . .796835) /note = “documented modABC operon” lux- + Operon (1635056 . . .1635481) /note = “predicted operon” a.pk0018.b10 /note = “ordered genescontained in the operon: b1549 ” lux-a.pk0022.h4 + Operon (816267 . . .818970) /note = “documented moaABCDE operon” lux-a.pk027.d12 + Operon(1879936 . . . 1881021) /note = “predicted operon” /note = “orderedgenes contained in the operon: b1800 ” lux-a.pk033.a4 + Operon (4213057. . . 4217911) /note = “documented aceBAK operon” lux-a.pk037.e6 +Operon (2232053 . . . 2234520) /note = “predicted operon” /note =“ordered genes contained in the operon: b2146 yeiA ” lux-a.pk041.h8 +Operon (2493599 . . . 2494585) /note = “predicted operon” /note =“ordered genes contained in the operon: b2378 ” lux-a.pk047.e8 − Operoncomplement(2239830 . . . 2241672) /note = “predicted operon” /note =“ordered genes contained in the operon: yeiB folE ” lux-a.pk052.h2 −Operon complement(3748758 . . . 3749498) /note = “predicted operon”/note = “ordered genes contained in the operon: yiaT ” lux-a.pk059.c12 −Operon complement(3944752 . . . 3945590) /note = “predicted operon”/note = “ordered genes contained in the operon: yifA pssR ”lux-a.pk066.c9 + Operon (3500404 . . . 3502421) /note = “predictedoperon” /note = “ordered genes contained in the operon: yhfP yhfQ yhfR”lux-a.pk072.f7 − Operon complement(1577657 . . . 1580581) /note =“predicted operon” /note = “ordered genes contained in the operon: b1497b1498” lux-a.pk078.e3 + Operon (2680877 . . . 2682076) /note =“predicted operon” /note = “ordered genes contained in the operon: b2550” lux-a.pk087.c1 − Operon complement(1234932 . . . 1236464) /note =“predicted operon” /note = “ordered genes contained in the operon: ycgB”lux-a.pk0001.b6 − Operon complement(4268628 . . . 4271450) /note =“documented uvrA operon” lux-a.pk007.e12 + Operon (4248534 . . .4249862) /note = “predicted operon” /note = “ordered genes contained inthe operon: yjbI ” lux-a.pk0018.c7 Operon complement(2245083 . . .2246552) /note = “predicted operon” /note = “ordered genes contained inthe operon: lysP ” lux-a.pk0022.h5 + Operon (2363915 . . . 2371298)/note = “predicted operon” /note = “ordered genes contained in theoperon: b2253 b2254 b2255 b2256 b2257 b2258 ” lux-a.pk027.e11 − Operoncomplement( 1762958 . . . 1766709) /note = “predicted operon” /note =“ordered genes contained in the operon: b1685 b1686 b1687 ”lux-a.pk033.a6 + Operon (3676830 . . . 3677978) /note = “predictedoperon” /note = “ordered genes contained in the operon: kdgK ”lux-a.pk037.e9 Operon complement(2097884 . . . 2099290) /note =“predicted operon” /note = “ordered genes contained in the operon: gnd ”lux-a.pk042.a6 − Operon complement(4228938 . . . 4229210) /note =“predicted operon” /note = “ordered genes contained in the operon: yjbD”lux-a.pk047.f2 − Operon complement(280053 . . . 281207) /note =“predicted operon” /note = “ordered genes contained in the operon: yagA” lux-a.pk052.h9 − Operon complement(2769861 . . . 2770706) /note =“predicted operon” /note = “ordered genes contained in the operon: b2638b2639” lux-a.pk060.a10 + Operon (3432844 . . . 3435531) /note =“predicted operon” /note = “ordered genes contained in the operon: fmutrkA ” lux-a.pk066.d11 + Operon (4411838 . . . 44134780 /note =“predicted operon” /note = “ordered genes contained in the operon: aidB” lux-a.pk072.g1 + Operon (3483757 . . . 3484389) /note = “documentedcrp operon” lux-a.pk078.f11 + Operon (3491648 . . . 3496838) /note =“documented nirBDC- cysG operon” lux-a.pk087.c4 − Operoncomplement(1232399 . . . 1233940) /note = “predicted operon” /note =“ordered genes contained in the operon: nhaB” lux-a.pk0001.c2 − Operoncomplement(1694486 . . . 1695076) /note = “predicted operon” /note =“ordered genes contained in the operon: gusR” lux-a.pk007.e7 + Operon(2837547 . . . 2840437) /note = “documented ascFB operon”lux-a.pk0018.d2 − Operon complement(605488 . . . 606606) /note =“predicted operon” /note = “ordered genes contained in the operon: b0581” lux- + Operon (2088214 . . . 2095247) /note = “documented a.pk0023.c11hisGDCBHAFI operon” lux-a.pk027.e3 − Operon complement(344890 . . .345561) /note = “predicted operon” /note = “ordered genes contained inthe operon: b0328 ” lux-a.pk033.c5 + Operon (1808223 . . . 1808825)/note = “predicted operon” /note = “ordered genes contained in theoperon: b1728 ” lux-a.pk037.f1 − Operon complement(2474714 . . .2475649) /note = “documented dsdC operon” lux-a.pk042.b3 − Operoncomplement(1838807 . . . 1839433) /note = “predicted operon” /note =“ordered genes contained in the operon: b1758” lux-a.pk047.f7 + Operon(2861616 . . . 2863035) /note = “predicted operon” /note = “orderedgenes contained in the operon: b2738 b2739” lux-a.pk053.b5 − Operoncomplement(464836 . . . 466536) /note = “predicted operon” /note =“ordered genes contained in the operon: ybaE ” lux-a.pk060.a6 + Operon(2775136 . . . 2775803) /note = “predicted operon” /note = “orderedgenes contained in the operon: b2645 b2646 ” lux-a.pk066.f11 − Operoncomplement(820765 . . . 823720) /note = “predicted operon” /note =“ordered genes contained in the operon: b0788 b0789 b0790 ”lux-a.pk072.g5 + Operon (1418389 . . . 1421668) /note = “predictedoperon” /note = “ordered genes contained in the operon: b1357 b1358b1359 b1360 b1361 b1362” lux-a.pk079.b10 − Operon complement(177001 . .. 179153) /note = “predicted operon” /note = “ordered genes contained inthe operon: yadS yadT pfs ” lux-a.pk087.c6 − Operon complement(2992482 .. . 2993114) /note = “predicted operon” /note = “ordered genes containedin the operon: b2855 b2856” lux-a.pk0001.c7 + Operon (2857783 . . .2858439) /note = “predicted operon” /note = “ordered genes contained inthe operon: b2734 ” lux-a.pk007.f11 − Operon complement(4429669 . . .4430643) /note = “predicted operon” /note = “ordered genes contained inthe operon: ytfF” lux-a.pk0018.d7 − Operon complement(3077663 . . .3079654) /note = “predicted operon” /note = “ordered genes contained inthe operon: tktA ” lux-a.pk023.c3 + Operon (3632372.3633523) /note =“predicted operon” /note = “ordered genes contained in the operon: yhiM” lux-a.pk027.e7 + Operon (997713 . . . 1003880) /note = “predictedoperon” /note = “ordered genes contained in the operon: b0939 b0940b0941 b0942 b0943 ycbF ” lux-a.pk033.d5 + Operon (4100373 . . . 4101077)/note = “predicted operon” /note = “ordered genes contained in theoperon: yiiM ” lux-a.pk037.f5 − Operon complement(2908778 . . . 2909361)/note = “predicted operon” /note = “ordered genes contained in theoperon: chpA chpR ” lux-a.pk042.b7 + Operon (3237584 . . . 3238828)/note = “predicted operon” /note = “ordered genes contained in theoperon: ygjU ” lux-a.pk047.g9 − Operon complement(2083726 . . . 2085090)/note = “predicted operon” /note = “ordered genes contained in theoperon: yeeF ” lux-a.pk053.b8 + Operon (3841591 . . . 3843357) /note =“predicted operon” /note = “ordered genes contained in the operon: yicP” lux-a.pk060.b12 − Operon complement(1325791 . . . 1327136) /note =“predicted operon” /note = “ordered genes contained in the operon: btuRyciK ” lux-a.pk066.g9 − Operon complement(1889349 . . . 1891259) /note =“predicted operon” /note = “ordered genes contained in the operon:b1808” lux-a.pk073.a11 − Operon complement(439426 . . . 440567) /note =“documented xseB-ispA operon” lux-a.pk079.e5 − Operon complement(346081. . . 347667) /note = “predicted operon” /note = “ordered genescontained in the operon: b0330 ” lux-a.pk087.d4 + Operon (252005 . . .253161) /note = “predicted operon” /note = “ordered genes contained inthe operon: yafN yafO yafP ” lux- − Operon complement(2591092 . . .2594757) /note = “predicted a.pk0001.d10 operon” /note = “ordered genescontained in the operon: b2473 b2474 b2475 ” lux-a.pk008.c11 − Operoncomplement(2420669 . . . 2421559) /note = “predicted operon” /note =“ordered genes contained in the operon: b2305 ” lux-a.pk0018.f1 + Operon(980270 . . . 982117) /note = “predicted operon” /note = “ordered genescontained in the operon: ycbB ” lux-a.pk0023.c6 + Operon (774376 . . .778255) /note = “documented tolQRAB operon” lux-a.pk027.f6 − Operoncomplement(691097 . . . 692640) /note = “predicted operon” /note =“ordered genes contained in the operon: b0659 b0660 ” lux-a.pk033.e12 −Operon complement(2112524 . . . 2116426) /note = “predicted operon”/note = “ordered genes contained in the operon: b2043 b2044 b2045 ”lux-a.pk037.h4 − Operon complement(4343258 . . . 4344904) /note =“documented fumB operon” lux-a.pk042.c7 + Operon (3416786 . . . 3418859)/note = “predicted operon” /note = “ordered genes contained in theoperon: yhdW yhdX ” lux-a.pk048.c7 + Operon (384399 . . . 387870) /note= “predicted operon” /note = “ordered genes contained in the operon:b0365 b0366 b0367 b0368 ” lux-a.pk053.d10 + Operon (4010643 . . .4012904) /note = “documented metE operon” lux-a.pk060.b6 + Operon(4349421 . . . 4349935) /note = “predicted operon” /note = “orderedgenes contained in the operon: yjdI yjdJ ” lux-a.pk067.b10 − Operoncomplement(2254105 . . . 2255355) /note = “predicted operon” /note =“ordered genes contained in the operon: yeiM ” lux-a.pk073.d7 − Operoncomplement( 1990897 . . . 1992663) /note = “predicted operon” /note =“ordered genes contained in the operon: uvrC ” lux-a.pk079.f10 − Operoncomplement(4452185 . . . 4453183) /note = “predicted operon” /note =“ordered genes contained in the operon: fbp” lux-a.pk087.e2 + Operon(2278652 . . . 2280412) /note = “predicted operon” /note = “orderedgenes contained in the operon: yejH ” lux- − Operon complement(59687 . .. 63264) /note = “predicted a.pk0001.f10 operon” /note = “ordered genescontained in the operon: yabO hepA ” lux-a.pk008.e12 + Operon (71351 . .. 72115) /note = “predicted operon” /note = “ordered genes contained inthe operon: yabI ” lux- + Operon (2890237 . . . 2892794) /note =“predicted operon” a.pk0018.f11 /note = “ordered genes contained in theoperon: b2765 b2766 b2767 b2768” lux-a.pk0023.d2 + Operon (15445 . . .16557) /note = “predicted operon” /note = “ordered genes contained inthe operon: yi81_1 ” lux-a.pk027.h4 − Operon complement(4590931 . . .4592292) /note = “predicted operon” /note = “ordered genes contained inthe operon: yjiZ ” lux-a.pk033.e2 + Operon (471822 . . . 473476) /note =“predicted operon” /note = “ordered genes contained in the operon: glnKamtB ” lux-a.pk037.h9 − Operon complement(904963 . . . 906012) /note =“predicted operon” /note = “ordered genes contained in the operon: b0868” lux-a.pk042.c8 + Operon (3208422 . . . 3212529) /note = “documentedrpsU- dnaG-rpoD operon” lux-a.pk048.d11 − Operon complement(1844989 . .. 1846032) /note = “documented selD operon” lux-a.pk053.d5 + Operon(4414530 . . . 4415279) /note = “predicted operon” /note = “orderedgenes contained in the operon: yjfP ” lux-a.pk060.e5 + Operon (3344219 .. . 3345879) /note = “predicted operon” /note = “ordered genes containedin the operon: ptsN b3205 ptsO ” lux-a.pk067.d11 − Operoncomplement(2715511 . . . 2716548) /note = “predicted operon” /note =“ordered genes contained in the operon: yfiFIf lux-a.pk073.f10 + Operon(3637741 . . . 3638175) /note = “predicted operon” /note = “orderedgenes contained in the operon: uspA ” lux-a.pk079.f4 + Operon (234798 .. . 235538) /note = “predicted operon” /note = “ordered genes containedin the operon: b0213 ” lux-a.pk087.f10 − Operon complement(4308686 . . .4309621) /note = “predicted operon” /note = “ordered genes contained inthe operon: yjcXII lux-a.pk0001.f2 − Operon complement(2426077 . . .2429677) /note = “documented dedD-cvpA-purF-ubiX operon”lux-a.pk009.a4 + Operon (3579494 . . . 3581811) /note = “predictedoperon” /note = “ordered genes contained in the operon: yhhZ b3443insA_5 insB_5” lux-a.pk0018.g6 + Operon (2415080 . . . 2416621) /note =“predicted operon” /note = “ordered genes contained in the operon: b2298” lux-a.pk0023.e3 + Operon (3223875 . . . 3225308) /note = “predictedoperon” /note = “ordered genes contained in the operon: ygjI ”lux-a.pk028.g6 − Operon complement(3663810 . . . 3665210) /note =“predicted operon” /note = “ordered genes contained in the operon: gadA”lux-a.pk033.e6 − Operon complement(3825087 . . . 3826292) /note =“predicted operon” /note = “ordered genes contained in the operon: gltS”lux-a.pk038.a4 − Operon complement(3623310 . . . 3628232) /note =“predicted operon” /note = “ordered genes contained in the operon: yhhJyhiG yhiI ” lux-a.pk042.d9 + Operon (1864932 . . . 1866866) /note =“predicted operon” /note = “ordered genes contained in the operon: b1783” lux-a.pk048.g4 + Operon (3029318 . . . 3030835) /note = “predictedoperon” /note = “ordered genes contained in the operon: b2888 ”lux-a.pk053.d8 − Operon complement(2523950 . . . 2524876) /note =“predicted operon” /note = “ordered genes contained in the operon: b2409” lux-a.pk060.e8 − Operon complement(740298 . . . 741779) /note =“predicted operon” /note = “ordered genes contained in the operon:b0709” lux-a.pk067.f8 − Operon complement(1921389 . . . 1922993) /note =“predicted operon” /note = “ordered genes contained in the operon: b1839b1840 b1841 ” lux-a.pk073.f3 − Operon complement(1515906 . . . 1516870)/note = “predicted operon” /note = “ordered genes contained in theoperon: b1447 b1448” lux-a.pk079.g6 − Operon complement(988377 . . .989579) /note = “documented pncB operon” lux-a.pk087.g3 − Operoncomplement(3475544 . . . 3476134) /note = “predicted operon” /note =“ordered genes contained in the operon: slyD ” lux- + Operon (484985 . .. 485632) /note = “predicted operon” /note a.pk0002.b12 = “ordered genescontained in the operon: acrR ” lux-a.pk009.a9 − Operoncomplement(232597 . . . 233955)/note = “predicted operon” /note =“ordered genes contained in the operon: dniR ” lux-a.pk0018.h2 + Operon(3782887 . . . 3785724) /note = “predicted operon” /note = “orderedgenes contained in the operon: yibO yibP ” lux-a.pk023.f2 − Operoncomplement(2030406 . . . 2031524) /note = “predicted operon” /note =“ordered genes contained in the operon: yedJ b1963 ” lux-a.pk029.b2 +Operon (3972208 . . . 3978309) /note = “predicted operon” /note =“ordered genes contained in the operon: yifH yifI yifJ rffT rffM ”lux-a.pk033.f3 + Operon (945094 . . . 947882) /note = “predicted operon”/note = “ordered genes contained in the operon: ycaD b0899 ”lux-a.pk038.b10 + Operon (1036963 . . . 1041138) /note = “documentedappCBA operon” lux-a.pk042.e10 − Operon complement(3464797 . . .3467490) /note = “predicted operon” /note = “ordered genes contained inthe operon: yheB ” lux-a.pk049.a4 − Operon complement( 1946774 . . .1948546) /note = “predicted operon” /note = “ordered genes contained inthe operon: aspS” lux-a.pk053.e9 − Operon complement(2077555 . . .2078613) /note = “predicted operon” /note = “ordered genes contained inthe operon: yeeA ” lux-a.pk060.g1 − Operon complement(1823979 . . .1830006) /note = “predicted operon” /note = “ordered genes contained inthe operon: b1744 b1745 b1746 b1747 b1748 ” lux-a.pk068.a3 − Operoncomplement(926697 . . . 930185) /note = “documented cydCD operon”lux-a.pk073.h2 + Operon (3646158 . . . 3648292) /note = “documentedarsRBC operon” lux-a.pk079.h9 − Operon complement(1532989 . . . 1533882)/note = “predicted operon” /note = “ordered genes contained in theoperon: yddE ” lux-a.pk088.c3 − Operon complement(264844 . . . 266191)/note = “predicted operon” /note = “ordered genes contained in theoperon: b0250 b0251 ” lux-lacZ complement(360473 . . . 365529)lux-a.pk009.b2 − Operon complement(1397745 . . . 1402604) /note =“predicted operon” /note = “ordered genes contained in the operon: ogtydaH b1337 b1338” lux- − Operon complement(3065360 . . . 3066100) /note= “predicted a.pk0019.a12 operon” /note = “ordered genes contained inthe operon: yggE ” lux-a.pk0023.h6 − Operon complement(1797417 . . .1800594) /note = “documented thrS-infC-rpmI-rplT operon” lux-a.pk029.b8− Operon complement(3487903 . . . 3489257) /note = “predicted operon”/note = “ordered genes contained in the operon: pabA fic yhfG ”lux-a.pk033.f6 + Operon (2380733 . . . 2381944) /note = “predictedoperon” /note = “ordered genes contained in the operon: b2269 ”lux-a.pk038.b6 + Operon (601182 . . . 602558) /note = “predicted operon”/note = “ordered genes contained in the operon: pheP ” lux-a.pk042.g1 +Operon (2898614 . . . 2901396) /note = “predicted operon” /note =“ordered genes contained in the operon: + ygcE ” lux-a.pk049.a8 + Operon(2556791 . . . 2558086) /note = “predicted operon” /note = “orderedgenes contained in the operon: b2442 ” lux-a.pk053.g7 − Operoncomplement(2180055 . . . 2180801) /note = “predicted operon” /note =“ordered genes contained in the operon: b2101 ” lux-a.pk061.a5 − Operoncomplement(3838176 . . . 3841494)/note = “predicted operon”/note =“ordered genes contained in the operon: yicM yicN yicO ” lux-a.pk068.b3− Operon complement(114407 . . . 117549) /note = “predicted operon”/note = “ordered genes contained in the operon: b0105 hofC hofB ppdD”lux-a.pk074.c11 + Operon (3555900 . . . 3557498) /note = “predictedoperon” /note = “ordered genes contained in the operon: yhgB ”lux-a.pk080.d1 + Operon (209679 . . . 212266) /note = “predicted operon”/note = “ordered genes contained in the operon: IdcC b0187 ”lux-a.pk088.c3 − Operon complement(264844 . . . 266191) /note =“predicted operon” /note = “ordered genes contained in the operon: b0250b0251 ” lux-a.pk0002.b7 − Operon complement(273325 . . . 274341) /note =“predicted operon” /note = “ordered genes contained in the operon:yi52_1 ” lux-a.pk009.c11 − Operon complement(3080896 . . . 3081816)/note = “predicted operon”/note = “ordered genes contained in theoperon: speB ” lux-a.pk0019.a8 + Operon (1896421 . . . 1898049) /note =“predicted operon” /note = “ordered genes contained in the operon: b1815” lux-a.pk0024.a3 + Operon (1244383 . . . 1244823) /note = “predictedoperon” /note = “ordered genes contained in the operon: b1196 ”lux-a.pk029.c10 − Operon complement(1550422 . . . 1550784) /note =“predicted operon” /note = “ordered genes contained in the operon: b1477” lux-a.pk033.g10 − Operon complement(1566978 . . . 1568513) /note =“predicted operon” /note = “ordered genes contained in the operon: xasA” lux-a.pk038.c2 + Operon (1599514 . . . 1605313) /note = “predictedoperon” /note = “ordered genes contained in the operon: b1513 b1514b1515 b1516 b1517 b1518” lux-a.pk043.c1 − Operon complement(2929887 . .. 2931710) /note = “documented fucAO operon” lux-a.pk049.d8 − Operoncomplement(5683 . . . 6459) /note = “predicted operon” /note = “orderedgenes contained in the operon: yaaA ” lux-a.pk054.a6 − Operoncomplement(4350778 . . . 4352295) /note = “documented lysU operon”lux-a.pk061.b6 − Operon complement(1815172 . . . 1819643) /note =“documented celABCDF operon” lux-a.pk068.b8 + Operon (1473162 . . .1475474) /note = “predicted operon” /note = “ordered genes contained inthe operon: ydbD ” lux-a.pk074.d9 + Operon (1903658 . . . 1904278) /note= “predicted operon” /note = “ordered genes contained in the operon:b1821 ” lux-a.pk080.e12 + Operon (243543 . . . 244121) /note =“predicted operon” /note = “ordered genes contained in the operon: gmhA” lux-a.pk088.d5 − Operon complement(3105038 . . . 3107233) /note =“documented speC operon” lux-lacZ complement(360473 . . . 365529)lux-a.pk009.d6 − Operon complement(642780 . . . 643190) /note =“predicted operon” /note = “ordered genes contained in the operon: rnk ”lux-a.pk0019.a9 + Operon (3484440 . . . 3486530) /note = “predictedoperon” /note = “ordered genes contained in the operon: yhfK ”lux-a.pk0024.b6 + Operon (122092 . . . 129336) /note = “documented pdhR-aceEF-lpdA operon” lux-a.pk029.c11 + Operon (4506526 . . . 4507122)/note = “predicted operon” /note = “ordered genes contained in theoperon: b4285 ” lux-a.pk033.g11 + Operon (1289465 . . . 1290478) /note =“predicted operon” /note = “ordered genes contained in the operon: hnr ”lux-a.pk038.c7 + Operon (2609941 . . . 2612802) /note = “predictedoperon” /note = “ordered genes contained in the operon: b2491 b2492 ”lux-a.pk043.e11 − Operon complement(2885601 . . . 2889921) /note =“documented cysJIH operon” lux-a.pk049.g7 − Operon complement(1886085 .. . 1887770) /note = “predicted operon” /note = “ordered genes containedin the operon: fadD ” lux-a.pk054.c10 − Operon complement(3317629 . . .3319272) /note = “predicted operon” /note = “ordered genes contained inthe operon: yhbX ” lux-a.pk061.b7 + Operon (180884 . . . 182308 /note =“predicted operon” /note = “ordered genes contained in the operon: htrA” lux-a.pk068.b9 + Operon (467520 . . . 471641 /note = “predictedoperon” /note = “ordered genes contained in the operon: b0447 mdlA mdlB” lux-a.pk074.e3 − Operon complement(4594719 . . . 4596971) /note =“predicted operon” /note = “ordered genes contained in the operon: mdoB” lux-a.pk080.g3 + Operon (655780 . . . 656340) /note = “predictedoperon” /note = “ordered genes contained in the operon: ybeG ”lux-a.pk088.e1 − Operon complement(1932863 . . . 1934338) /note =“documented zwf operon” lux-a.pk0002.e4 + Operon (1735868 . . . 1736893)/note = “documented purR operon” lux-a.pk009.d11 − Operoncomplement(4363050 . . . 4364351) /note = “predicted operon” /note =“ordered genes contained in the operon: dcuA ” lux- + Operon (2621064 .. . 2623130) /note = “documented ppk a.pk0019.c11 operon”lux-a.pk0024.c7 − Operon complement(592551 . . . 594666) /note =“predicted operon” /note = “ordered genes contained in the operon: b0570b0571 ” lux-a.pk029.c5 − Operon complement(266408 . . . 267244) /note =“predicted operon” /note = “ordered genes contained in the operon: b0252” lux-a.pk033.g2 − Operon complement(2131512 . . . 2135.265) /note =“predicted operon” /note = “ordered genes contained in the operon: b2060b2061 b2062” lux-a.pk038.d9 − Operon complement(65855 . . . 70048) /note= “documented araBAD operon” lux-a.pk043.f11 + Operon (4200898 . . .4202223) /note = “predicted operon” /note = “ordered genes contained inthe operon: hydG ” lux-a.pk049.h9 + Operon (3352267.3359575) /note =“documented gltBDF operon” lux-a.pk054.c3 − Operon complement(2729620 .. . 2732193) /note = “predicted operon” /note = “ordered genes containedin the operon: clpB” lux-a.pk061.c3 + Operon (202560 . . . 208608) /note= “predicted operon” /note = “ordered genes contained in the operon:lpxA lpxB rnhB dnaE ” lux-a.pk068.h2 + Operon (103155 . . . 106456)/note = “documented ftsQAZ operon” lux-a.pk074.f1 + Operon (2550372 . .. 2552144) /note = “predicted operon” /note = “ordered genes containedin the operon: amiA hemF” lux-a.pk081.b5 + Operon (3728760 . . .3733786) /note = “documented xylFGHR operon” lux-a.pk088.g2 + Operon(882896 . . . 884128) /note = “predicted operon” /note = “ordered genescontained in the operon: b0842 ” lux-lacZ complement(360473 . . .365529) lux-a.pk009.f3 − f176; This 176 aa ORF is 45 pct identical (2gaps) to 172 residues of an approx. 184 aa protein FIMF_ECOLI SW: P08189lux- − Operon complement(4061182 . . . 4066856) /note = “predicteda.pk0019.d12 operon” /note = “ordered genes contained in the operon:yihO yihO yihP yihQ ” lux-a.pk0024.e1 − Operon complement(2854476 . . .2854829) /note = “predicted operon” /note = “ordered genes contained inthe operon: ygbA ” lux-a.pk029.c5 + Operon (2766686 . . . 2767507) /note= “predicted operon” /note = “ordered genes contained in the operon:b2633 ” lux-a.pk033.g7 + Operon (2001895 . . . 2004101) /note =“documented fliDST operon” lux-a.pk038.e11 + Operon (3785854 . . .3786687) /note = “predicted operon” /note = “ordered genes contained inthe operon: yibQ ” lux-a.pk043.f6 − Operon complement(3040509 . . .3041168) /note = “predicted operon” /note = “ordered genes contained inthe operon: b2899 ” lux-a.pk050.a11 − Operon complement(4437449 . . .4438792) /note = “predicted operon” /note = “ordered genes contained inthe operon: ytfL ” lux-a.pk054.e11 − Operon complementer(3711690 . . .3713909) /note = “predicted operon” /note = “ordered genes contained inthe operon: bisCII lux-a.pk061.d8 − Operon complement(3148833 . . .3149999) /note = “documented exbDB operon” lux-a.pk068.h4 + Operon(3180566 . . . 3181339) /note = “predicted operon” /note = “orderedgenes contained in the operon: ygiE ” lux-a.pk074.g9 − Operoncomplement(4000900 . . . 4002326) /note = “predicted operon” /note =“ordered genes contained in the operon: rarDyigI ” lux-a.pk081.b6 +Operon (879950 . . . 881152) /note = “predicted operon” /note = “orderedgenes contained in the operon: dacC ” lux-a.pk088.h3 + Operon (122092 .. . 129336) /note = “documented pdhR-aceEF- pdA operon”lux-a.pk0002.g5 + Operon (2342885 . . . 2346534) /note = “documentednrdAB operon” lux-a.pk009.g3 − Operon complement(2181736 . . . 2183321)/note = “predicted operon” /note = “ordered genes contained in theoperon: b2103 b2104” lux-a.pk0019.e3 − Operon complement(4012721 . . .4013719) /note = “predicted operon” /note = “ordered genes contained inthe operon: ” lux-a.pk0024.e6 + Operon (893007 . . . 897152) /note =“documented potFGHI operon” lux-a.pk029.c9 + Operon (89634 . . . 103153)/note = “predicted operon” /note = “ordered genes contained in theoperon: yabB yabC ftsL ftsI murE murF mraY murD ftsW murG murC ddlB ”lux-a.pk034.a11 − Operon complement(2116702 . . . 2119576) /note =“predicted operon” /note = “ordered genes contained in the operon: b2046b2047” lux-a.pk038.f1 + Operon (1830452 . . . 1831258) /note =“predicted operon” /note = “ordered genes contained in the operon: xthA” lux-a.pk043.g5 − Operon complement(1957304 . . . 1957876) /note =“predicted operon” /note = “ordered genes contained in the operon: b1875” lux-a.pk050.a12 − Operon complement(4338298 . . . 4339206) /note =“documented melR operon” lux-a.pk054.e5 − Operon complement(3858976 . .. 3861491)/note = “predicted operon”/note = “ordered genes contained inthe operon: glvG glvB glvC” lux-a.pk061.e9 − Operon complement(2980519 .. . 2982146) /note = “predicted operon” /note = “ordered genes containedin the operon: kduD kduI ” lux-a.pk069.a4 − Operon complement(3026544 .. . 3028966) /note = “predicted operon” /note = “ordered genes containedin the operon: b2886 b2887” lux-a.pk074.h10 + Operon (4109895 . . .4110335) /note = “predicted operon” /note = “ordered genes contained inthe operon: yiiR ” lux-a.pk081.c2 − Operon complement(4457474 . . .4457938) /note = “predicted operon” /note = “ordered genes contained inthe operon: nrdG” lux-a.pk089.a11 + Operon (1094746 . . . 1096052) /note= “predicted operon” /note = “ordered genes contained in the operon:b1028 b1029” lux-a.pk0003.a2 + Operon (349236 . . . 353816) /note =“predicted operon” /note = “ordered genes contained in the operon: b0333b0334 b0335 ” lux-a.pk009.g7 − FecR protein lux-a.pk0019.e4 + Operon(1303788 . . . 1304792) /note = “predicted operon” /note = “orderedgenes contained in the operon: oppF ” lux-a.pk0024.f5 + Operon (832293 .. . 835433) /note = “predicted operon” /note = “ordered genes containedin the operon: dinG ybiB ” lux-a.pk029.e7 − Operon complement(1852120 .. . 1852878) /note = “predicted operon” /note = “ordered genes containedin the operon: b1770 ” lux-a.pk034.b8 + Operon (4126252 . . . 4129847)/note = “documented metBL operon” lux-a.pk038.f3 − Operoncomplement(303719 . . . 309250) /note = “predicted operon” /note =“ordered genes contained in the operon: b0289 b0290 b0291 b0292 ”lux-a.pk043.h1 + Operon (2383874 . . . 2384851) /note = “predictedoperon” /note = “ordered genes contained in the operon: b2271 ”lux-a.pk050.b9 − Operon complement(360473 . . . 365529) /note =“documented lacAYZ operon” lux-a.pk054.f7 + Operon (1057307 . . .1061621) /note = “documented torCAD operon” lux-a.pk061.f10 − Operoncomplement(2612840 . . . 2613901) /note = “predicted operon” /note =“ordered genes contained in the operon: b2493 ” lux-a.pk069.a6 − Operoncomplement(4372207 . . . 4373235) /note = “predicted operon” /note =“ordered genes contained in the operon: yjeKft lux-a.pk075.a10 + Operon(4609980 . . . 4611053) /note = “predicted operon” /note = “orderedgenes contained in the operon: yjjU ” lux-a.pk081.e3 − Operoncomplement(2024345 . . . 2026054) /note = “predicted operon” /note =“ordered genes contained in the operon: b1956 ” lux-a.pk089.c4 + Operon(434858 . . . 436331) /note = “predicted operon” /note = “ordered genescontained in the operon: b0417 b0418 ” lux-a.pk0003.c1 + Operon (1216509. . . 1218074) /note = “predicted operon” /note = “ordered genescontained in the operon: b1168 ” lux- + Operon (738224 . . . 740148)/note = “predicted operon” /note = a.pk0010.a12 “ordered genes containedin the operon: ybgA phrB ” lux-a.pk0019.f8 + Operon (970975 . . .971868) /note = “predicted operon” /note = “ordered genes contained inthe operon: b0919 ” lux-a.pk0024.f7 − Operon complement(4555923 . . .4558261) /note = “predicted operon” /note = “ordered genes contained inthe operon: iadA yjiG yjiH ” lux-a.pk029.g2 − Operon complement(50380 .. . 51222) /note = “predicted operon” /note = “ordered genes containedin the operon: apaH ” lux-a.pk034.b9 − Operon complement(3843403 . . .3844794) /note = “documented uhpT operon” lux-a.pk038.g11 + Operon(2212886 . . . 2213617) /note = “predicted operon” /note = “orderedgenes contained in the operon: yehV ” lux-a.pk044.b6 − Operoncomplement(3451145 . . . 3453035) /note = “predicted operon” /note =“ordered genes contained in the operon: pinO yheD ” lux-a.pk050.d9 −Operon complement(2759372 . . . 2763174) /note = “predicted operon”/note = “ordered genes contained in the operon: b2627 b2628”lux-a.pk054.h8 + Operon (195785 . . . 200360)/note = “predicted operon”/note = “ordered genes contained in the operon: cdsA yaeL b0177 ”lux-a.pk061.f4 + Operon (4382971 . . . 4383596) /note = “predictedoperon” /note = “ordered genes contained in the operon: yjeN yjeO ”lux-a.pk069.a8 − Operon complement(3673920 . . . 3675995) /note =“predicted operon” /note = “ordered genes contained in the operon: yhjF” lux-a.pk075.c5 + Operon (2192320 . . . 2194353) /note = “predictedoperon” /note = “ordered genes contained in the operon: metG ”lux-a.pk081.f12 + Operon (3031085 . . . 3031633) /note = “predictedoperon” /note = “ordered genes contained in the operon: b2889 ”lux-a.pk089.c6 − Operon complement(4091029 . . . 4095029) /note =“documented rhaBAD operon” lux-a.pk0003.d1 − Operon complement(1246919 .. . 1250091) /note = “predicted operon” /note = “ordered genes containedin the operon: ycgC b1199 b1200 ” lux-a.pk0010.b4 + Operon (1923464 . .. 1924806) /note = “predicted operon” /note = “ordered genes containedin the operon: b1843 b1844” lux-a.pk0019.g1 + Operon 2010524 . . .2010802 /note = “predicted operon” /note = “ordered genes contained inthe operon: b1936 ” lux-a.pk0024.g2 − Operon complement(394354 . . .395511) /note = “predicted operon” /note = “ordered genes contained inthe operon: yaiH ” lux-a.pk029.g4 − Operon complement(4112149 . . .4113159) /note = “predicted operon” /note = “ordered genes contained inthe operon: glpX ” lux-a.pk034.d1 − Operon complement(4519695 . . .4523371) /note = “predicted operon” /note = “ordered genes contained inthe operon: yjhG yjhH yjhI ” lux-a.pk038.h1 − Operon complement( 1654771. . . 1655517) /note = “predicted operon” /note = “ordered genescontained in the operon: b1585 ” lux-a.pk044.b7 − Operoncomplement(4097072 . . . 4098106) /note = “documented rhaT operon”lux-a.pk050.e1 − Operon complement(2906051 . . . 2907688) /note =“predicted operon” /note = “ordered genes contained in the operon: pyrG” lux-a.pk055.a12 − Operon complement(3275497 . . . 3276306) /note =“predicted operon” /note = “ordered genes contained in the operon: agaR” lux-a.pk061.f5 − Operon complement(3924173 . . . 3924631) /note =“documented asnC operon” lux-a.pk069.d3 − Operon complement(4125658 . .. 4125975) /note = “documented metJ operon” lux-a.pk075.d9 + Operon3220238 . . . 3223812 /note = “documented ebgAC operon” lux-a.pk081.f3 −Operon complement(2570177 . . . 2573897) /note = “predicted operon”/note = “ordered genes contained in the operon: cchA eutI b2459 b2460b2461 b2462 ” lux-a.pk089.e2 − Operon complement(2624715 . . . 2626958)/note = “predicted operon” /note = “ordered genes contained in theoperon: b2503 ” lux- + Operon (4022578 . . . 4024818) /note = “predictedoperon” a.pk0003.e12 /note = “ordered genes contained in the operon:yigC ubiB ” lux-a.pk0010.c7 − Operon complement(3750593 . . .3752058)/note = “predicted operon”/note = “ordered genes contained inthe operon: yiaV yiaW” lux-a.pk0019.g2 − Operon complement(2654556 . . .2657487) /note = “predicted operon” /note = “ordered genes contained inthe operon: yfhJ fdx hscA yfhE ” lux-a.pk0024.g3 + Operon (2032043 . . .2032777) /note = “predicted operon” /note = “ordered genes contained inthe operon: b1964 b1965” lux-a.pk030.b3 − Operon complement(4272339 . .. 4272689) /note = “predicted operon” /note = “ordered genes containedin the operon: yjcB ” lux-a.pk034.d3 − Operon complement(2858490 . . .2859287) /note = “predicted operon” /note = “ordered genes contained inthe operon: b2735 ” lux-a.pk038.h6 − Operon complement(2441911 . . .2446793) /note = “predicted operon” /note = “ordered genes contained inthe operon: b2325 b2326 yfcA mepA aroC yfcB ” lux-a.pk044.d10 − Operoncomplement(3668922 . . . 3669524) /note = “predicted operon” /note =“ordered genes contained in the operon: yhjB” lux-a.pk050.e10 + Operon(4439959 . . . 4445812) /note = “predicted operon” /note = “orderedgenes contained in the operon: ytfM ytfN ytfP ” lux-a.pk055.a3 + Operon(250898 . . . 251953) /note = “predicted operon” /note = “ordered genescontained in the operon: ” lux-a.pk061.h3 − Operon complement(489334 . .. 490036) /note = “predicted operon” /note = “ordered genes contained inthe operon: ybaM priC ” lux-a.pk069.d8 + Operon (424235 . . . 429700)/note = “documented queA-tgt- yajC-secD-secF operon” lux-a.pk075.f11 −Operon complement(2478658 . . . 2481359) /note = “predicted operon”/note = “ordered genes contained in the operon: emrY emrK ”lux-a.pk081.g10 + Operon (1773611 . . . 1776371) /note = “predictedoperon” /note = “ordered genes contained in the operon: ydiF b1695 ”lux-a.pk089.e4 − Operon complement(2563501 . . . 2570070) /note =“predicted operon” /note = “ordered genes contained in the operon: b2451eutH eutG eutJ eutE cchB ” lux-a.pk0003.f1 − Operon complement(1252308 .. . 1255175) /note = “predicted operon” /note = “ordered genes containedin the operon: b1202 ” lux-a.pk0010.d6 − Operon complement(2181736 . . .2183321) /note = “predicted operon”/note = “ordered genes contained inthe operon: b2103 b2104 ” lux- − Operon complement(805221 . . . 806504)/note = “predicted a.pk0020.b12 operon” /note = “ordered genes containedin the operon: ybhC ” lux-a.pk0024.g8 − Operon complement(3463886 . . .3464362) /note = “predicted operon” /note = “ordered genes contained inthe operon: bfr ” lux-a.pk030.b8 + Operon (208621 . . . 209580) /note =“documented accA operon” lux-a.pk034.d6 + Operon (4501566 . . . 4503973)/note = “predicted operon” /note = “ordered genes contained in theoperon: yjhB yjhC ” lux-a.pk039.a5 − Operon complement(437539 . . .439401) /note = “predicted operon” /note = “ordered genes contained inthe operon: b0420 ” lux-a.pk044.f8 − Operon complement(4620670 . . .4622358) /note = “predicted operon” /note = “ordered genes contained inthe operon: lplA smp ” lux-a.pk050.e4 + Operon (1958086 . . . 1959819)/note = “predicted operon” /note = “ordered genes contained in theoperon: argS ” lux-a.pk055.a4 − Operon complement(899067 . . . 902957)/note = “documented artPIQMJ operon” lux-a.pk062.a12 − Operoncomplement(1431108 . . . 1431698) /note = “predicted operon” /note =“ordered genes contained in the operon: b1374” lux-a.pk069.f11 − Operoncomplement(3393963 . . . 3396015) /note = “predicted operon” /note =“ordered genes contained in the operon: cafA yhdE ” lux-a.pk075.f4 +Operon (47246 . . . 49631) /note = “predicted operon” /note = “orderedgenes contained in the operon: yabF kefC ” lux-a.pk081.h2 + Operon(2852361 . . . 2854439) /note = “predicted operon” /note = “orderedgenes contained in the operon: fhlA ” lux-a.pk089.e7 + Operon (3208422 .. . 3212529) /note = “documented rpsU- dnaG-rpoD operon” lux-a.pk0004.a2− Operon complement(2493070 . . . 2493312) /note = “predicted operon”/note = “ordered genes contained in the operon: b2377 ” lux- − Operoncomplement(411831 . . . 416176) /note = “predicted a.pk0010.f10 operon”/note = “ordered genes contained in the operon: sbcC sbcD ”lux-a.pk0020.b4 + Operon (4105132 . . . 4106094) /note = “predictedoperon” /note = “ordered genes contained in the operon: pfkA ”lux-a.pk0024.h2 + Operon (2808791 . . . 2809321) /note = “predictedoperon” /note = “ordered genes contained in the operon: emrR ”lux-a.pk030.d3 + Operon (2042885 . . . 2050036) /note = “predictedoperon” /note = “ordered genes contained in the operon: b1978 ”lux-a.pk034.e1 + Operon (253467 . . . 254202) /note = “predicted operon”/note = “ordered genes contained in the operon: b0235 prfH ”lux-a.pk039.a6 − Operon complement(4553059 . . . 4553889) /note =“predicted operon” /note = “ordered genes contained in the operon: yjiC”lux-a.pk044.f9 + Operon (4339489 . . . 4342368) /note = “documentedmelAB operon” lux-a.pk050.e9 − Operon complement(910405 . . . 913043)/note = “predicted operon” /note = “ordered genes contained in theoperon: b0872 b0873 ” lux-a.pk055.b2 − Operon complement(930308 . . .931273) /note = “predicted operon” /note = “ordered genes contained inthe operon: trxB ” lux-a.pk062.b4 − Operon complement(2513663 . . .2515969) /note = “predicted operon” /note = “ordered genes contained inthe operon: yfeA ” lux-a.pk069.f4 + Operon (3866983 . . . 3868068) /note= “predicted operon” /note = “ordered genes contained in the operon:yidS ” lux-a.pk075.f5 − Operon complement(2447248 . . . 2453021) /note =“predicted operon” /note = “ordered genes contained in the operon: b2332b2333 b2334 b2335 b2336 b2337 b2338 ” lux-a.pk081.h3 − Operoncomplement(4577638 . . . 4580618) /note = “documented hsdMS operon”lux-a.pk089.f1 − Operon complement(3144871 . . . 3145706) /note =“predicted operon” /note = “ordered genes contained in the operon: b2999b3000” lux-lacZ complement(360473 . . . 365529) lux- − Operoncomplement(3261327 . . . 3264706) /note = a.pk0010.g10 “documentedtdcABC operon” lux-a.pk0020.c2 + Operon (320832 . . . 323677) /note =“predicted operon” /note = “ordered genes contained in the operon: b0306b0307 b0308 ” lux-a.pk0024.h3 + Operon (2990116 . . . 2991492) /note =“predicted operon” /note = “ordered genes contained in the operon: b2852” lux-a.pk030.f10 − Operon complement(4067055 . . . 4067981) /note =“predicted operon” /note = “ordered genes contained in the operon: yihR” lux-a.pk034.f5 − Operon complement(1417789 . . . 1418265) /note =“predicted operon” /note = “ordered genes contained in the operon:b1356” lux-a.pk039.b6 + Operon (547838 . . . 550555) /note = “predictedoperon” /note= “ordered genes contained in the operon: b0519 b0520 ybcF” lux-a.pk044.g3 + Operon (1421806 . . . 1424004) /note = “predictedoperon” /note = “ordered genes contained in the operon: trkG b1364 b1365b1366” lux-a.pk050.f6 − Operon complement(2643033 . . . 2650307) /note =“predicted operon” /note = “ordered genes contained in the operon: b2519b2520” lux-a.pk055.b3 + Operon (179237 . . . 180754) /note = “predictedoperon” /note = “ordered genes contained in the operon: dgt ”lux-a.pk062.c6 − Operon complement(602639 . . . 603886) /note =“predicted operon” /note = “ordered genes contained in the operon: ybdG” lux-a.pk069.f6 + Operon (4324713 . . . 4327816) /note = “predictedoperon” /note = “ordered genes contained in the operon: yjdA yjcZ ”lux-a.pk075.g1 − Operon complement(2519613 . . . 2522898) /note =“predicted operon” /note = “ordered genes contained in the operon: xapRxapB xapA ” lux-a.pk081.h7 − Operon complement(2224529 . . . 2225290)/note = “predicted operon” /note = “ordered genes contained in theoperon: yohF” lux-a.pk089.g1 + Operon (3776681 . . . 3778644) /note -”predicted operon” /note = “ordered genes contained in the operon: lctRlctD ” lux-a.pk0004.c1 + Operon (1848884 . . . 1849900) /note =“documented ansA operon” lux- + Operon (3225442 . . . 3228880) /note =“predicted operon” a.pk0010.g12 /note = “ordered genes contained in theoperon: ygjJ ygjK ” lux-a.pk0020.c3 − Operon complement(2638706 . . .2640864) /note = “predicted operon” /note = “ordered genes contained inthe operon: gcpE yfgA ” lux- − Operon complement(3132887 . . . 3134386)/note = “predicted a.pk0025.c11 operon” /note = “ordered genes containedin the operon: pitB” lux-a.pk030.f2 − Operon complement(1061773 . . .1062998) /note = “predicted operon” /note = “ordered genes contained inthe operon: yccD cbpA ” lux-a.pk034.g4 + Operon (106557 . . . 107474)/note = “predicted operon” /note = “ordered genes contained in theoperon: lpxC ” lux-a.pk039.d1 + Operon (339389 . . . 341731) /note =“predicted operon” /note = “ordered genes contained in the operon: b0323b0324 ” lux-a.pk044.h4 + Operon (3535122 . . . 3537344) /note =“predicted operon” /note = “ordered genes contained in the operon: yhgF” lux-a.pk050.g1 + Operon (3150251 . . . 3151438) /note = “documentedmetC operon” lux-a.pk055.d10 − Operon complement(3363337 . . . 3364353)/note = “predicted operon” /note = “ordered genes contained in theoperon: yi52_1 ” lux-a.pk062.e11 + Operon (4055987 . . . 4057762) /note= “predicted operon” /note = “ordered genes contained in the operon:yihK ” lux-a.pk069.g7 + Operon (190 . . . 5020) /note = “documentedthrLABC operon” lux-a.pk075.h9 − Operon cpmplement(858436 . . . 859251)/note = “predicted operon” /note = “ordered genes contained in theoperon: b0822 ” lux-a.pk081.h8 − Operon complement(1145234 . . .1145857) /note = “predicted operon” /note = “ordered genes contained inthe operon: yceFft lux-a.pk090.a2 + Operon (1312044 . . . 1312682) /note= “predicted operon” /note = “ordered genes contained in the operon:yciD ” lux-lacZ complement(360473 . . . 365529) lux-a.pk0011.a2 + Operon(539783 . . . 542257) /note = “predicted operon” /note = “ordered genescontained in the operon: b0513 b0514 ” lux- − Operon complement(296605 .. . 301797) /note = “predicted a.pk0020.d10 operon” /note = “orderedgenes contained in the operon: b0282 b0283 b0284 b0285 b0286 ”lux-a.pk0025.c9 − Operon complement(2238648 . . . 2239688) /note =“documented galS operon” lux-a.pk030.f8 − Operon complement(4535227 . .. 4537078) /note = “predicted operon” /note = “ordered genes containedin the operon: yjhT yjhA ” lux-a.pk034.h2 − Operon complement(720279 . .. 723637) /note = “documented kdpDE operon” lux-a.pk039.d10 − Operoncomplement(1411555 . . . 1415410) /note = “predicted operon” /note =“ordered genes contained in the operon: ydaC lar recT recE ”lux-a.pk045.c12 − Operon complement(3068185 . . . 3071711) /note =“predicted operon” /note = “ordered genes contained in the operon: fbapgk epd ” lux-a.pk050.g10 − Operon complement(2227458 . . . 2228405)/note = “predicted operon” /note = “ordered genes contained in theoperon: yohI” lux-a.pk055.e12 − Operon complement(1447100 . . . 1449373)/note = “predicted operon” /note = “ordered genes contained in theoperon: tynA” lux-a.pk062.e7 − Operon complement(3171520 . . . 3175926)/note = “predicted operon” /note = “ordered genes contained in theoperon: parE yqiA icc yqiB b3034 ” lux-a.pk070.a4 − Operoncomplement(3904481 . . . 3909153) /note = “documented pstSCAB-phoUoperon” lux-a.pk076.c10 + Operon (22391 . . . 27227) /note = “documentedileS-lspA-lytB operon” lux-a.pk082.a8 + Operon (972760 . . . 980009)/note = “predicted operon” /note = “ordered genes contained in theoperon: smtA mukF mukE mukB ” lux-a.pk090.g5 − Operon complement(3043178. . . 3043921) /note = “predicted operon” /note = “ordered genescontained in the operon: ygfF” lux- − Operon complement(1492172 . . .1493236) /note = “predicted a.pk0004.e11 operon” /note = “ordered genescontained in the operon: b1422 ” lux-a.pk0011.a6 + Operon (4619338 . . .4620669) /note = “predicted operon” /note = “ordered genes contained inthe operon: yjjJ ” lux- + Operon (5234 . . . 5530) /note = “predictedoperon” /note = a.pk0020.d11 “ordered genes contained in the operon:b0005 ” lux- − Operon complement(2409459 . . . 2410632) /note =“predicted a.pk0025.d10 operon” /note = “ordered genes contained in theoperon: b2293 b2294” lux-a.pk030.g7 − Operon complement(10643 . . .11356) /note = “predicted operon” /note = “ordered genes contained inthe operon: b0011 ” lux-a.pk034.h6 + Operon (2160898 . . . 2163020)/note = “documented baeSR operon” lux-a.pk039.e8 + Operon (4008666 . . .4009565) /note = “predicted operon” /note = “ordered genes contained inthe operon: yigM ” lux-a.pk045.c2 − Operon complement(2699761 . . .2702083) /note = “documented rnc-era-recO operon” lux-a.pk050.g2 −Operon complement(550750 . . . 552323) /note = “documented purEK operon”lux-a.pk055.g12 − Operon complement(1877427 . . . 1877972) /note =“predicted operon” /note = “ordered genes contained in the operon: b1796b1797” lux-a.pk062.f10 − Operon complement(4294798 . . . 4296945) /note= “documented fdhF operon” lux-a.pk070.b7 − Operon complement(2223064 .. . 2223675) /note = “predicted operon” /note = “ordered genes containedin the operon: yohC ” lux-a.pk076.c5 − Operon complement(3795866 . . .3805725) /note = “documented rfaQGPSBIJYZK operon” lux-a.pk082.b4 −Operon complement(3396024 . . . 3398784) /note = “documented mreBCDoperon” lux-a.pk089.g1 + Operon (3776681 . . . 3778644) /note =“predicted operon” /note = “ordered genes contained in the operon: lctRlctD ” lux-lacZ complement(360473 . . . 365529) lux-a.pk0011.d6 − Operoncomplement(542485 . . . 545587) /note = “predicted operon” /note =“ordered genes contained in the operon: b0515 b0516 b0517 ” lux- −Operon complement(3981965 . . . 3983620) /note = “predicted a.pk0020.g10operon” /note = “ordered genes contained in the operon: aslA ” lux- −Operon complement(1274402 . . . 1276841) /note = a.pk0025.e12“documented narXL operon” lux-a.pk030.h2 + Operon (3234934 . . .3235938) /note = “predicted operon” /note = “ordered genes contained inthe operon: ygjR ” lux-a.pk035.a10 − Operon complement(747144 . . .751401) /note = “predicted operon” /note = “ordered genes contained inthe operon: b0716 b0717 b0718 ” lux-a.pk039.f11 + Operon (269466 . . .270978) /note = “predicted operon” /note = “ordered genes contained inthe operon: b0255 tra8_1 ” lux-a.pk045.d11 − Operon complement(3508698 .. . 3509763) /note = “predicted operon” /note = “ordered genes containedin the operon: yhfY yhfZ ” lux-a.pk050.g3 − Operon complement(276980 . .. 279099) /note = “predicted operon” /note = “ordered genes contained inthe operon: yagC b0263 insB_1 insA_2 ” lux-a.pk055.g3 − Operoncomplement(149715 . . . 152854) /note = “predicted operon” /note =“ordered genes contained in the operon: yadC yadK yadL yadM ”lux-a.pk062.h4 + Operon (632809 . . . 633969) /note = “predicted operon”/note = “ordered genes contained in the operon: b0600 ” lux-a.pk070.c1 +Operon (440773 . . . 442221) /note = “predicted operon” /note = “orderedgenes contained in the operon: b0423 ” lux-a.pk076.c8 + Operon (319451 .. . 320305) /note = “predicted operon” /note = “ordered genes containedin the operon: b0305 ” lux-a.pk082.b5 − Operon complement(3868065 . . .3869402) /note = “predicted operon” /note = “ordered genes contained inthe operon: yidT” lux-a.pk090.a2 + Operon (1312044 . . . 1312682) /note= “predicted operon” /note = “ordered genes contained in the operon:yciD ” lux-a.pk0004.f7 − Operon complement(2034816 . . . 2036893) /note= “predicted operon” /note = “ordered genes contained in the operon:b1968 b1969” lux-a.pk0011.d7 + Operon (70387 . . . 71265) /note =“documented araC operon” lux-a.pk0020.g3 + Operon (770678 . . . 773404)/note = “documented cydAB operon” lux-a.pk0025.e2 + Operon (4569935 . .. 4571500) /note = “predicted operon” /note = “ordered genes containedin the operon: yjiT ” lux-a.pk031.a12 + Operon (4266993 . . . 4267706)/note = “predicted operon” /note = “ordered genes contained in theoperon: yjbP ” lux-a.pk035.b7 − Operon complement(3348330 . . . 3350660)/note = “predicted operon” /note = “ordered genes contained in theoperon: arcB ” lux-a.pk039.g11 + Operon (2923370 . . . 2924218) /note =“predicted operon” /note = “ordered genes contained in the operon: b2794” lux-a.pk045.d6 + Operon (1143671 . . . 1144045) /note = “predictedoperon” /note = “ordered genes contained in the operon: b1085 ”lux-a.pk050.g7 + Operon (1475639 . . . 1480225) /note = “predictedoperon” /note = “ordered genes contained in the operon: b1408 b1409b1410 b1411” lux-a.pk055.h3 + Operon (3089126 . . . 3091935) /note =“predicted operon” /note = “ordered genes contained in the operon: yggJgshB b2948 b2949 ” lux-a.pk063.d5 − Operon complement(3545619 . . .3550106) /note = “documented malPQ operon” lux-a.pk070.c11 − Operoncomplement(1041253 . . . 1048555) /note = “predicted operon” /note =“ordered genes contained in the operon: yccC b0982 b0983 b0984 b0985b0986 ” lux-a.pk076.e8 − Operon complement(3056686 . . . 3057345) /note= “predicted operon” /note = “ordered genes contained in the operon:rpiA ” lux-a.pk082.c8 + Operon (3698192 . . . 3699463) /note =“predicted operon” /note = “ordered genes contained in the operon: yhjV” lux-a.pk090.g5 − Operon complement(3043178 . . . 3043921) /note =“predicted operon” /note = “ordered genes contained in the operon: ygfF”lux-a.pk005.b11 − Operon complement(2752029 . . . 2752785) /note =“predicted operon” /note = “ordered genes contained in the operon: b2618b2619” lux-a.pk0011.h8 − Operon (420210 . . . 421583) /note = “predictedoperon” /note = “ordered genes contained in the operon: b0402 ”lux-a.pk0020.g6 − Operon complement(587205 . . . 592401) /note =“documented nfrBA operon” lux-a.pk0025.f1 + Operon (1892829 . . .1894772) /note = “predicted operon” /note = “ordered genes contained inthe operon: pabB yeaB ” lux-a.pk031.a3 + Operon (3405238 . . . 3407588)/note = “documented panF- prmA operon” lux-a.pk035.c7 − Operoncomplement(3383856 . . . 3387041) /note = “predicted operon” /note =“ordered genes contained in the operon: yhcP yhcQ b3242 ” lux-a.pk039.h3− Operon complement(2868278 . . . 2870843) /note = “predicted operon”/note = “ordered genes contained in the operon: b2745 ygbB b2747 b2748 ”lux-a.pk045.f3 + Operon (1463416 . . . 1465974) /note = “predictedoperon” /note = “ordered genes contained in the operon: ydbA_1 ”lux-a.pk050.h3 + Operon (4483786 . . . 4485968) /note = “predictedoperon” /note = “ordered genes contained in the operon: yjgP yjgQ ”lux-a.pk055.h9 + Operon (784856 . . . 785908) /note = “documented aroGoperon” lux-a.pk063.d9 + Operon (705316 . . . 706980) /note = “predictedoperon” /note = “ordered genes contained in the operon: glnS ”lux-a.pk070.d2 − Operon complement(859397 . . . 862761) /note =“predicted operon” /note = “ordered genes contained in the operon: b0823b0824 ” lux-a.pk076.f2 + Operon (1777641 . . . 1779363) /note =“predicted operon” /note = “ordered genes contained in the operon: b1697b1698” lux-a.pk082.d4 + Operon (4552145 . . . 4552918) /note =“predicted operon” /note = “ordered genes contained in the operon: uxuR” lux-a.pk005.b6 + Operon (2151891 . . . 2160901) /note = “predictedoperon” /note = “ordered genes contained in the operon: b2074 b2075b2076 b2077” lux-a.pk0012.c3 + Operon (3478926 . . . 3482073) /note =“predicted operon” /note = “ordered genes contained in the operon: yheSyheT yheU ” lux- − Operon complement(2500010 . . . 2506262) /note =“predicted a.pk0020.h11 operon” /note = “ordered genes contained in theoperon: b2383 b2384 b2385 b2386 b2387 ” lux-a.pk0025.f3 + Operon(2194494 . . . 2201931) /note = “predicted operon” /note = “orderedgenes contained in the operon: molR molR molR yehI ” lux-a.pk031.c11 −Operon complement(3146992 . . . 3147486) /note = “predicted operon”/note = “ordered genes contained in the operon: b3002 ”lux-a.pk035.g11 + Operon (3101031 . . . 3102386) /note = “predictedoperon” /note = “ordered genes contained in the operon: mutY b2962”lux-a.pk039.h5 − Operon complement(2469097 . . . 2471266) /note =“predicted operon” /note = “ordered genes contained in the operon: b2354b2355 b2356 b2357 b2358 ” lux-a.pk045.g1 − Operon complement(4346893 . .. 4349240) /note = “predicted operon” /note = “ordered genes containedin the operon: yjdG yjdH ” lux-a.pk051.b1 − Operon complement(1158585 .. . 1160774) /note = “predicted operon” /note = “ordered genes containedin the operon: fhuE” lux-a.pk056.a11 + Operon (571689 . . . 572956)/note = “predicted operon” /note = “ordered genes contained in theoperon: b0547 b0548 b0549 b0550 ” lux-a.pk063.e1 − Operoncomplement(1181006 . . . 1184817) /note = “documented potABCD operon”lux-a.pk070.e2 + Operon (624108 . . . 628520) /note = “documentedentCEBA operon” lux-a.pk076.f8 − Operon complement(3640010 . . .3642812) /note = “predicted operon” /note = “ordered genes contained inthe operon: yhiQ prlC ” lux-a.pk082.d5 − Operon complement(2706774 . . .2708032) /note = “documented rpoE-rseA operon” lux-a.pk005.e12 + Operon(4435285 . . . 4435785) /note = “predicted operon” /note = “orderedgenes contained in the operon: b4215 ” lux-a.pk0012.c7 − Operoncomplement(3472315 . . . 3474089) /note = “predicted operon” /note =“ordered genes contained in the operon: yheL b3344 yheN b3346 ”lux-a.pk0020.h5 − Operon complement(1841855 . . . 1844984) /note =“predicted operon” /note = “ordered genes contained in the operon: b1762topB ” lux-a.pk0025.f5 + Operon (164730 . . . 167264) /note = “predictedoperon” /note = “ordered genes contained in the operon: mrcB ”lux-a.pk031.c7 − Operon complement(3031677 . . . 3034226) /note =“documented prfB-lysS operon” lux-a.pk035.h9 − Operon complement(1863750. . . 1864496) /note = “predicted operon” /note = “ordered genescontained in the operon: b1782 ” lux-a.pk040.a11 + Operon (1710793 . . .1712295) /note = “predicted operon” /note = “ordered genes contained inthe operon: b1634 ” lux-a.pk045.g4 − Operon complement(2833196 . . .2835448) /note = “predicted operon” /note = “ordered genes contained inthe operon: hypF” lux-a.pk051.b11 − Operon complement(3112567 . . .3117128) /note = “predicted operon” /note = “ordered genes contained inthe operon: b2973 b2974” lux-a.pk056.b3 − Operon complement(2948657 . .. 2956906) /note = “predicted operon” /note = “ordered genes containedin the operon: recD recB ptr” lux-a.pk063.e8 + Operon (1807404 . . .1808072) /note = “predicted operon” /note = “ordered genes contained inthe operon: b1727 ” lux-a.pk070.e4 + Operon (2589267 . . . 2590754)/note = “predicted operon” /note = “ordered genes contained in theoperon: yffB dapE ” lux-a.pk077.a11 − Operon complement(608682 . . .611717) /note = “documented fepA-entD operon” lux-a.pk082.e8 + Operon (4028751 . . . 4032033) /note = “predicted operon” /note = “ordered genescontained in the operon: pepQ yigZ trkH ” lux-a.pk005.f6 + Operon(3602024 . . . 3602879) /note = “predicted operon” /note = “orderedgenes contained in the operon: yhhF b3466 ” lux-a.pk0012.e3 + Operon(3540803 . . . 3541681) /note = “predicted operon” /note = “orderedgenes contained in the operon: yhgA ” lux- + Operon (3285731 . . .3290073) /note = “predicted operon” a.pk0021.a10 /note = “ordered genescontained in the operon: yraI yraJ yraK ” lux- − Operoncomplement(4159749 . . . 4160849) /note = “predicted a.pk0025.g11operon” /note = “ordered genes contained in the operon: trmA ”lux-a.pk031.d1 − Operon complement(1396798 . . . 1397550) /note =“documented fnr operon” lux-a.pk036.a5 − Operon complement(4586446 . . .4588864) /note = “predicted operon” /note = “ordered genes contained inthe operon: yjiX yjiY ” lux-a.pk040.b5 − Operon complement(3913181 . . .3920080) /note = “documented atpIBEFHAGDC operon” lux-a.pk046.a5 +Operon (1846861 . . . 1848717) /note = “predicted operon” /note =“ordered genes contained in the operon: sppA ” lux-a.pk051.c11 − Operoncomplement(1607253 . . . 1608704) /note = “documented uxaB operon”lux-a.pk056.b4 − Operon complement(1588878 . . . 1590466) /note =“documented hipBA operon” lux-a.pk063.g7 + Operon (1741481 . . .1742854) /note = “predicted operon” /note = “ordered genes contained inthe operon: ydhE ” lux-a.pk070.f11 + Operon (3834580 . . . 3835764)/note = “predicted operon” /note = “ordered genes contained in theoperon: yicK ” lux-a.pk077.a3 − Operon complement(3130469 . . . 3131972)/note = “predicted operon” /note = “ordered genes contained in theoperon: b2984 b2985” lux-a.pk082.g4 + Operon (108279 . . . 110984) /note= “predicted operon” /note = “ordered genes contained in the operon:secA ” lux-a.pk005.g10 + Operon (1486256 . . . 1487695) /note =“predicted operon” /note = “ordered genes contained in the operon: aldA” lux- + Operon (45807 . . . 47138) /note = “predicted operon” /note =a.pk0012.f11 “ordered genes contained in the operon: yaaU ”lux-a.pk0021.b6 − Operon complement(3427403 . . . 3429566) /note =“predicted operon” /note = “ordered genes contained in the operon: yrdBaroE yrdC yrdD ” lux-a.pk0025.g5 + Operon (738224 . . . 740148) /note =“predicted operon” /note = “ordered genes contained in the operon: ybgAphrB” lux-a.pk031.e7 + Operon (27293 . . . 28207) /note = “predictedoperon” /note = “ordered genes contained in the operon: yaaF ”lux-a.pk036.b6 − Operon complement(4428899 . . . 4429561) /note =“predicted operon” /note = “ordered genes contained in the operon: b4209” lux-a.pk040.b7 − Operon complement(1582231 . . . 1584510) /note =“predicted operon” /note = “ordered genes contained in the operon: b1501” lux-a.pk046.b5 − Operon complement(3044188 . . . 3048687) /note =“documented gcvTHP operon” lux-a.pk051.d10 − Operon complement(4156969 .. . 4158303) /note = “predicted operon” /note = “ordered genes containedin the operon: udhA ” lux-a.pk056.c3 − Operon complement(332725 . . .333657) /note = “predicted operon” /note = “ordered genes contained inthe operon: b0316” lux-a.pk064.c10 − Operon complement(2945779 . . .2947122) /note = “predicted operon” /note = “ordered genes contained inthe operon: b2817 ” lux-a.pk070.g5 + Operon (4328080 . . . 4329582)/note = “documented proP operon” lux-a.pk077.a9 − Operoncomplement(229967 . . . 230881) /note = “predicted operon” /note =“ordered genes contained in the operon: yafC lux-a.pk082.h2 + Operon(1753722 . . . 1755134) /note = “predicted operon” /note = “orderedgenes contained in the operon: pykF ” lux-a.pk005.g2 + Operon (4049619 .. . 4050998) /note = “predicted operon” /note = “ordered genes containedin the operon: hemN ” lux-a.pk0015.d6 + Operon (1218824 . . . 1220344)/note = “predicted operon” /note = “ordered genes contained in theoperon: b1169 ” lux-a.pk0021.b8 + Operon (3928943 . . . 3930502) /note =“predicted operon” /note = “ordered genes contained in the operon: kup ”lux-a.pk0025.h1 + Operon (1785469 . . . 1786302) /note = “predictedoperon” /note = “ordered genes contained in the operon: ydiA ”lux-a.pk031.e9 + Operon (533140 . . . 535710) /note = “predicted operon”/note = “ordered genes contained in the operon: gcl gip ”lux-a.pk036.b9 + Operon (938651 . . . 939943) /note = “predicted operon”/note = “ordered genes contained in the operon: serS ” lux-a.pk040.c9 −Operon complement(583903 . . . 586131) /note = “documented envY-ompToperon” lux-a.pk046.c1 + Operon (535810 . . . 538311) /note = “predictedoperon” /note = “ordered genes contained in the operon: b0509 b0510b0511 ” lux-a.pk051.d5 + Operon (638168 . . . 640541) /note =“documented ahpCF operon” lux-a.pk056.d6 + Operon (1360767 . . .1364839) /note = “predicted operon” /note = “ordered genes contained inthe operon: aldH ordL goaG ” lux-a.pk064.c7 − Operon complement(1853015. . . 1859356) /note = “predicted operon” /note = “ordered genescontained in the operon: b1771 b1772 b1773 b1774 b1775 b1776 ”lux-a.pk070.g8 − Operon complement(408332 . . . 409243) /note =“predicted operon” /note = “ordered genes contained in the operon: yaiD”lux-a.pk077.b1 − Operon complement(751452 . . . 752018) /note =“predicted operon” /note = “ordered genes contained in the operon: ybgD” lux-a.pk083.a4 − Operon complement(505827 . . . 506306) /note =“predicted operon” /note = “ordered genes contained in the operon: b0481” lux-lacZ complement(360473 . . . 365529) lux-a.pk0016.c4 + Operon(819107 . . . 819811) /note = “predicted operon” /note = “ordered genescontained in the operon: b0786 ” lux-a.pk0021.c6 − Operoncomplement(1244902 . . . 1246599) /note = “predicted operon” /note =“ordered genes contained in the operon: treA” lux-a.pk0025.h5 − Operoncomplement(764376 . . . 765098) /note = “predicted operon” /note =“ordered genes contained in the operon: farR” lux-a.pk031.f10 + Operon(2261883 . . . 2263064) /note = “predicted operon” /note = “orderedgenes contained in the operon: yeiO ” lux-a.pk036.d10 + Operon (4156069. . . 4156986) /note = “documented oxyR operon” lux-a.pk040.d7 − Operoncomplement(2671836 . . . 2677387) /note = “predicted operon” /note =“ordered genes contained in the operon: b2544 b2545 b2546 b2547 b2548 ”lux-a.pk046.c12 + Operon (2282149 . . . 2284156) /note = “predictedoperon” /note = “ordered genes contained in the operon: yejL yejM ”lux-a.pk051.g11 − Operon complement(1722760 . . . 1724082) /note =“predicted operon” /note = “ordered genes contained in the operon: b1647b1648” lux-a.pk056.f2 + Operon (1631646 . . . 1632236) /note =“predicted operon” /note = “ordered genes contained in the operon: b1545” lux-a.pk064.f4 + Operon (3274643.3275442) /note = “predicted operon”/note = “ordered genes contained in the operon: sohA yhaV ”lux-a.pk071.a11 − Operon complement(906075 . . . 910272) /note =“predicted operon” /note = “ordered genes contained in the operon: b0869b0870 poxB ” lux-a.pk077.c5 + Operon (3884457 . . . 3885821) /note =“predicted operon” /note = “ordered genes contained in the operon: thdF” lux-a.pk083.d3 − Operon complement(2436962 . . . 2438140) /note =“predicted operon” /note = “ordered genes contained in the operon: b2322” lux-a.pk005.g3 − Operon complement(841019 . . . 841423) /note =“predicted operon” /note = “ordered genes contained in the operon: b0806” lux- − Operon complement(4333272 . . . 4334609) /note = “predicteda.pk0016.a11 operon” /note = “ordered genes contained in the operon:yjdD ” lux-a.pk0021.d4 + Operon (4133655 . . . 4134593) /note =“predicted operon” /note = “ordered genes contained in the operon: yijE” lux- + Operon (4448633 . . . 4452152) /note = “predicted operon”a.pk0026.a12 /note = “ordered genes contained in the operon: ytfR ytfSytfT yjfF ” lux-a.pk031.f7 + Operon (3735126 . . . 3737156) /note =“documented malS operon” lux-a.pk036.d6 − Operon complement(837753 . . .840754) /note = “predicted operon” /note = “ordered genes contained inthe operon: b0804 b0805 ” lux-a.pk040.e4 + Operon (3791614 . . .3795834) /note = “documented rfaDFCL operon” lux-a.pk046.c3 + Operon(2765725 . . . 2766594) /note = “predicted operon” /note = “orderedgenes contained in the operon: b2632 ” lux-a.pk051.h4 − Operoncomplement(2943058 . . . 2943864) /note = “predicted operon” /note =“ordered genes contained in the operon: b2812 ” lux-a.pk056.g9 − Operoncomplement(717485 . . . 719683) /note = “documented speF operon”lux-a.pk064.f8 + Operon (3058870 . . . 3064300) /note = “predictedoperon” /note = “ordered genes contained in the operon: sbm ygfD b2919b2920 ” lux-a.pk071.a4 − Operon complement(4051449 . . . 4055614) /note= “documented glnALG operon” lux-a.pk077.c7 + Operon (3416027 . . .3416248) /note = “predicted operon” /note = “ordered genes contained inthe operon: yhdV ” lux-a.pk083.e7 − Operon complement(1120784 . . .1121830) /note = “documented pyrC operon” lux-lacZ complement(360473 . .. 365529) lux-a.pk0016.g4 + Operon (2848670 . . . 2852287) /note =“documented hypABCDE operon” lux-a.pk0021.e5 + Operon (331595 . . .332683) /note = “predicted operon” /note = “ordered genes contained inthe operon: yahA ” lux-a.pk0026.b5 + Operon (3229306 . . . 3231324)/note = “predicted operon” /note = “ordered genes contained in theoperon: ygjL ” lux-a.pk031.g10 + Operon (1097070 . . . 1098047) /note =“predicted operon” /note = “ordered genes contained in the operon: b1033” lux-a.pk036.d7 − Operon complement(1665368 . . . 1666588) /note =“predicted operon” /note = “ordered genes contained in the operon: mlc”lux-a.pk040.e5 + Operon (4422696 . . . 4424135) /note = “documentedrpsF- priB-rpsR-rplI operon” lux-a.pk046.e6 − Operon complement(4085688. . . 4090404) /note = “predicted operon” /note = “ordered genescontained in the operon: frvR frvX frvB frvA ” lux-a.pk052.a4 − Operoncomplement(476291 . . . 477847) /note = “predicted operon” /note =“ordered genes contained in the operon: b0457 ” lux-a.pk056.h3 + Operon(1725861 . . . 1726268) /note = “predicted operon” /note = “orderedgenes contained in the operon: b1651 ” lux-a.pk064.g8 − Operoncomplement(2225343 . . . 2226859) /note = “predicted operon” /note =“ordered genes contained in the operon: yohG yohH ” lux-a.pk071.e1 +Operon (3244277 . . . 3245068) /note = “predicted operon” /note =“ordered genes contained in the operon: exuR ” lux-a.pk077.d11 + Operon(3291041 . . . 3294625) /note = “predicted operon” /note = “orderedgenes contained in the operon: yraM yraN yraO yraP ” lux-a.pk083.g7 +Operon (1532048 . . . 1532893) /note = “predicted operon” /note =“ordered genes contained in the operon: b1463 ” lux-a.pk006.b2 − Operoncomplement(2417861 . . . 2418505) /note = “predicted operon” /note =“ordered genes contained in the operon: b2301 ” lux-a.pk0016.h6 + Operon(8238 . . . 9191) /note = “predicted operon” /note = “ordered genescontained in the operon: talB ” lux-a.pk0021.e6 + Operon (1944176 . . .1944877) /note = “predicted operon” /note = “ordered genes contained inthe operon: yebB ” lux- − Operon complement(3875333 . . . 3877747) /note= “predicted a.pk0026.c12 operon” /note = “ordered genes contained inthe operon: gyrB ” lux-a.pk031.g7 − Operon complement(1020953 . . .1023571) /note = “predicted operon” /note = “ordered genes contained inthe operon: b0960 yccF ” lux-a.pk036.d9 − Operon complement(3250958 . .. 3251854) /note = “predicted operon” /note = “ordered genes containedin the operon: yhaJ ” lux-a.pk040.g11 + Operon (1321244 . . . 1324665)/note = “predicted operon” /note = “ordered genes contained in theoperon: b1266 yciO yciQ ” lux-a.pk046.e9 − Operon complement(184257 . .. 188650) /note = “predicted operon” /note = “ordered genes contained inthe operon: yaeI b0165 dapD glnD ” lux-a.pk052.a9 − Operoncomplement(4280832 . . . 4282792) /note = “predicted operon” /note =“ordered genes contained in the operon: yjcG yjcH ” lux-a.pk057.f5 −Operon complement(4458096 . . . 4460234) /note = “predicted operon”/note = “ordered genes contained in the operon: nrdD ” lux-a.pk064.h7 −Operon complement(2748136 . . . 2748729) /note = “predicted operon”/note = “ordered genes contained in the operon: grpE ” lux-a.pk071.f7 −Operon complement(2547666 . . . 2548592) /note = “predicted operon”/note = “ordered genes contained in the operon: b2431 ” lux-a.pk077.d9 +Operon (4628275 . . . 4630239) /note = “predicted operon” /note =“ordered genes contained in the operon: slt ” lux-a.pk083.h2 − Operoncomplement(1169741 . . . 1173187) /note = “predicted operon” /note =“ordered genes contained in the operon: mfd ” lux-lacZ complement(360473. . . 365529) lux-a.pk0017.a4 + Operon (142779 . . . 144472) /note =“predicted operon” /note = “ordered genes contained in the operon: yadGyadH ” lux-a.pk0021.e8 − Operon complement(4612249 . . . 4614634) /note= “predicted operon” /note = “ordered genes contained in the operon:yjjW yjjI ” lux-a.pk0026.c7 + Operon (2318063 . . . 2321271) /note =“predicted operon” /note = “ordered genes contained in the operon: atoSatoC ” lux-a.pk031.g8 − Operon complement(3129356 . . . 3130333) /note =“predicted operon” /note = “ordered genes contained in the operon: b2983” lux-a.pk036.e6 − Operon complement(3836802 . . . 3837620) /note =“predicted operon” /note = “ordered genes contained in the operon: nlpA” lux-a.pk040.g3 − Operon complement(3927224 . . . 3928744) /note =“predicted operon” /note = “ordered genes contained in the operon: yieN” lux-a.pk046.f11 + Operon (4014920 . . . 4016347) /note = “predictedoperon” /note = “ordered genes contained in the operon: yigN ”lux-a.pk052.b6 + Operon (3995596 . . . 3997758) /note = “documented uvrDoperon” lux-a.pk058.a11 − Operon complement(947883 . . . 948791) /note =“predicted operon” /note = “ordered genes contained in the operon: b0900” lux-a.pk064.h8 − Operon complement(1550852 . . . 1551892) /note =“predicted operon” /note = “ordered genes contained in the operon: b1478” lux-a.pk071.g2 − Operon complement(4585479 . . . 4586333) /note =“predicted operon” /note = “ordered genes contained in the operon: yjiAm lux-a.pk077.e4 − Operon complement(1596641 . . . 1598233) /note =“predicted operon” /note = “ordered genes contained in the operon: b1511” lux-a.pk085.a11 + Operon (2435970 . . . 2436965) /note = “predictedoperon” /note = “ordered genes contained in the operon: div ”lux-a.pk006.b4 − Operon complement(1313880 . . . 1314116) /note =“predicted operon” /note = “ordered genes contained in the operon: yciG”lux- − Operon complement(4413595 . . . 4413897) /note = “predicteda.pk0017.c10 operon” /note = “ordered genes contained in the operon:yjfN ” lux-a.pk0021.f6 − Operon complement(3215197 . . . 3216717) /note= “predicted operon” /note = “ordered genes contained in the operon: air” lux-a.pk0026.c8 + Operon (2496691 . . . 2500007) /note = “predictedoperon” /note = “ordered genes contained in the operon: b2380 b2381b2382 ” lux-a.pk031.g9 + Operon (358023 . . . 360370) /note =“documented cynTSX operon” lux-a.pk036.e8 − Operon complement(2455035 .. . 2458489) /note = “predicted operon” /note = “ordered genes containedin the operon: b2341 b2342” lux-a.pk040.g4 − Operon complement(3137731 .. . 3143155) /note = “documented hybGFEDCBA operon” lux-a.pk046.g7 −Operon complement(1355826 . . . 1357265) /note = “predicted operon”/note = “ordered genes contained in the operon: b1296 ” lux-a.pk052.b7 −Operon complement(1884888 . . . 1886015) /note = “predicted operon”/note = “ordered genes contained in the operon: rnd” lux-a.pk058.a9 +Operon (3963322 . . . 3963705) /note = “predicted operon” /note =“ordered genes contained in the operon: trxA ” lux-a.pk065.d9 − Operoncomplement(1878910 . . . 1879854) /note = “predicted operon” /note =“ordered genes contained in the operon: b1799” lux-a.pk071.g4 − Operoncomplement(3160760 . . . 3161497) /note = “predicted operon” /note =“ordered genes contained in the operon: plsC” lux-a.pk077.f10 + Operon(2991660 . . . 2991878) /note = “predicted operon” /note = “orderedgenes contained in the operon: b2853 ” lux-a.pk085.a5 + Operon (4254694. . . 4256700) /note = “documented lexA- dinF operon” lux-a.pk006.c9 +Operon (4381375 . . . 4382919) /note = “predicted operon” /note =“ordered genes contained in the operon: yjeM ” lux-a.pk0017.c7 + Operon(504138 . . . 505790) /note = “predicted operon” /note = “ordered genescontained in the operon: ushA ” lux- + Operon (458112 . . . 460466)/note = “predicted operon” /note a.pk0021.g11 = “ordered genes containedin the operon: lon ” lux-a.pk0026.d1 + Operon (113444 . . . 114487)/note = “predicted operon” /note = “ordered genes contained in theoperon: guaC ” lux-a.pk032.b10 + Operon (1262937 . . . 1268242) /note =“predicted operon” /note = “ordered genes contained in the operon: hemAprfA hemK b1213 ychA kdsA ” lux-a.pk036.h7 + Operon (182445 . . .183620) /note = “predicted operon” /note = “ordered genes contained inthe operon: yaeG ” lux-a.pk040.g6 − Operon complement(2434735 . . .2435871) /note = “predicted operon” /note = “ordered genes contained inthe operon: pdxB ” lux-a.pk047.a10 + Operon (2011251 . . . 2017535)/note = “documented fliFGHIJK operon” lux-a.pk052.c11 + Operon (1124785. . . 1126952) /note = “predicted operon” /note = “ordered genescontained in the operon: rimJ yceH ” lux-a.pk058.c1 − Operoncomplement(3253729 . . . 3255597) /note = “predicted operon” /note =“ordered genes contained in the operon: yhaN yhaO ” lux-a.pk065.e4 +Operon (3272923 . . . 3274494) /note = “predicted operon” /note =“ordered genes contained in the operon: yhaG ” lux-a.pk071.g6 − Operoncomplement(4454888 . . . 4455439) /note = “predicted operon” /note =“ordered genes contained in the operon: yjgA ” lux-a.pk077.f12 − Operoncomplement(2841059 . . . 2848458) /note = “documented hycABCDEFGHoperon” lux-a.pk085.c1 − Operon complement(944154 . . . 944780) /note =“predicted operon” /note = “ordered genes contained in the operon: ycaC” lux-a.pk006.e10 − Operon complement(2122545 . . . 2131419) /note =“predicted operon” /note = “ordered genes contained in the operon: yefDyefC yefB yefA b2054 b2055 b2056 b2057 wcaB b2059 ” lux-a.pk0017.c9 +Operon (3958292 . . . 3960313) /note = “predicted operon” /note =“ordered genes contained in the operon: rep ” lux-a.pk0022.a2 − Operoncomplement(2536692 . . . 2541548) /note = “documented cysPUWAM operon”lux- + Operon (3610600 . . . 3611187) /note = “predicted operon”a.pk0026.e10 /note = “ordered genes contained in the operon: yhhU ”lux-a.pk032.b2 + Operon (3281811 . . . 3284666) /note = “predictedoperon” /note = “ordered genes contained in the operon: agaB agaC agaDagaI ” lux-a.pk036.h9 + Operon (830095 . . . 831459) /note = “predictedoperon” /note = “ordered genes contained in the operon: rhlE ”lux-a.pk041.a10 − Operon complement(3780269 . . . 3781755) /note =“predicted operon” /note = “ordered genes contained in the operon: gpsAsecB ” lux-a.pk047.a11 + Operon (4016442 . . . 4021926) /note =“predicted operon” /note = “ordered genes contained in the operon: yigOyigP yigQ yigTa yigTb yigTc yigU yigW yigW ” lux-a.pk052.c5 − Operoncomplement(2574118 . . . 2576397) /note = “predicted operon” /note =“ordered genes contained in the operon: b2463 ” lux-a.pk058.c11 − Operoncomplement(1980578 . . . 1984151) /note = “documented araFG-b1899-araHoperon” lux-a.pk065.f11 − Operon complement(3376505 . . . 3377632)/note - ”predicted operon” /note = “ordered genes contained in theoperon: yhcM ” lux-a.pk072.a10 − Operon complement(149715 . . . 152854)/note = “predicted operon” /note = “ordered genes contained in theoperon: yadC yadK yadL yadM ” lux-a.pk077.f3 + Operon (3965531 . . .3972101) /note = “predicted operon” /note = “ordered genes contained inthe operon: rfe b3785 rffE rffD rffG rffH ” lux-a.pk085.f1 − Operoncomplement(54755 . . . 57109) /note = “predicted operon” /note =“ordered genes contained in the operon: imp” lux-a.pk006.f7 − Operoncomplement(3865689 . . . 3866939) /note = “predicted operon” /note =“ordered genes contained in the operon: yidR” lux-a.pk0017.d3 + Operon(4476036 . . . 4476452) /note = “predicted operon” /note = “orderedgenes contained in the operon: yjgD ” lux-a.pk0022.b1 − Operoncomplement(4025199 . . . 4028561) /note = “documented fadBA operon” lux-− Operon complement(78848 . . . 83708) /note = “documented a.pk0026.f11leuLABCD operon” lux-a.pk032.c3 − Operon complement(2744454 . . .2745815) /note = “predicted operon” /note = “ordered genes contained inthe operon: ffh ” lux-a.pk037.a3 + Operon (3857880 . . . 3858803) /note= “predicted operon” /note = “ordered genes contained in the operon:b3680” lux-a.pk041.a6 − Operon complement(640662 . . . 641090) /note =“predicted operon” /note = “ordered genes contained in the operon: b0607” lux-a.pk047.a6 − Operon complement(661975 . . . 663186) /note =“predicted operon” /note = “ordered genes contained in the operon: dacA” lux-a.pk052.d6 − Operon complement(3892901 . . . 3894238) /note =“predicted operon” /note = “ordered genes contained in the operon: yieG”lux-a.pk058.c9 − Operon complement(1187539 . . . 1189670) /note =“documented phoQP operon” lux-a.pk065.f9 + Operon (2135858 . . .2137507) /note = “predicted operon” /note = “ordered genes contained inthe operon: b2063 ” lux-a.pk072.b1 − Operon complement(1846149 . . .1846700) /note = “predicted operon” /note = “ordered genes contained inthe operon: ydjA ” lux-a.pk077.h3 − Operon complement(4534182 . . .4535162) /note = “predicted operon” /note = “ordered genes contained inthe operon: yjhS ” lux-a.pk085.g10 − Operon complement(1015762 . . .1017522) /note = “predicted operon” /note = “ordered genes contained inthe operon: b0955 ” lux-a.pk006.h1 + Operon (989845 . . . 992457) /note= “predicted operon” /note = “ordered genes contained in the operon:pepN ” lux-a.pk0017.e3 − Operon complement(2334813 . . . 2337440) /note= “documented gyrA operon” lux-a.pk0022.b3 − Operon complement(4188313 .. . 4193677) /note = “documented thiCEFGH operon” lux-a.pk0026.f3 +Operon (4276058 . . . 4277407) /note = “predicted operon” /note =“ordered genes contained in the operon: yjcD ” lux-a.pk032.d2 + Operon(2461032 . . . 2462090) /note = “predicted operon” /note = “orderedgenes contained in the operon: b2345 ” lux-a.pk037.c11 − Operoncomplement(824225 . . . 829878) /note = “predicted operon” /note =“ordered genes contained in the operon: b0792 b0793 b0794 b0795 ybiH ”lux-a.pk041.b3 − Operon complement(3334604 . . . 3337706) /note =“predicted operon” /note = “ordered genes contained in the operon: yrbByrbC b3193 yrbE b3195 ” lux-a.pk047.b4 + Operon (2735619 . . . 2736925)/note = “documented pheLA operon” lux-a.pk052.d7 + Operon (1732459 . . .1733274) /note = “predicted operon” /note = “ordered genes contained inthe operon: b1655 ” lux-a.pk058.e10 + Operon (1928905 . . . 1930083)/note = “predicted operon” /note = “ordered genes contained in theoperon: purT ” lux-a.pk065.g3 − Operon complement(2246757 . . . 2247638)/note = “predicted operon” /note = “ordered genes contained in theoperon: yeiE” lux-a.pk072.b6 + Operon (538371 . . . 539732) /note =“predicted operon” /note = “ordered genes contained in the operon: b0512” lux-a.pk078.b3 − Operon complement(383283 . . . 383693) /note =“predicted operon” /note = “ordered genes contained in the operon: b0364” lux-a.pk085.h9 + Operon (532235 . . . 533050) /note = “predictedoperon” /note = “ordered genes contained in the operon: b0506 ”lux-a.pk006.h2 − Operon complement(2595851 . . . 2597780) /note =“documented dapA-nlpB operon” lux-a.pk0017.e7 + Operon (3656862 . . .3661157) /note = “predicted operon” /note = “ordered genes contained inthe operon: yhiU yhiV ” lux-a.pk0022.b5 − Operon complement(4148026 . .. 4150677) /note = “predicted operon” /note = “ordered genes containedin the operon: ppc” lux- + Operon (940269 . . . 944119) /note =“documented dmsABC a.pk0026.g10 operon” lux-a.pk032.d7 + Operon (432226. . . 434780) /note = “predicted operon” /note = “ordered genescontained in the operon: ybaD ribD ribH nusB ” lux-a.pk037.c3 − Operoncomplement(658474 . . . 661435) /note = “predicted operon” /note =“ordered genes contained in the operon: lipA ybeF lipB” lux-a.pk041.d4 +Operon (1737935 . . . 1739146) /note = “predicted operon” /note =“ordered genes contained in the operon: ydhC ” lux-a.pk047.c5 − Operoncomplement(3322642 . . . 3325305) /note = “documented ftsJ-hflB operon”lux-a.pk052.e11 − Operon complement(2712459 . . . 2713385) /note =“predicted operon” /note = “ordered genes contained in the operon: yfiE” lux-a.pk058.f2 − Operon complement(1942370 . . . 1944000) /note =“documented ruvBA operon” lux-a.pk065.g6 + Operon (3155664 . . .3156593) /note = “predicted operon” /note = “ordered genes contained inthe operon: + ” lux-a.pk072.b8 + Operon (3018561 . . . 3022207) /note =“predicted operon” /note = “ordered genes contained in the operon: b2880b2881 ” lux-a.pk078.c2 − Operon complement(288525 . . . 289529) /note =“documented argF operon” lux-a.pk086.a5 − Operon complement(4302191 . .. 4304188) /note = “predicted operon” /note = “ordered genes containedin the operon: yjcS ” lux-a.pk007.a10 − Operon complement(2338437 . . .2342189) /note = “predicted operon” /note = “ordered genes contained inthe operon: yfaL” lux- − Operon complement(2633619 . . . 2635415) /note= “predicted a.pk0017.f11 operon” /note = “ordered genes contained inthe operon: b2510 b2511” lux-a.pk0022.c1 + Operon (334504 . . . 339313)/note = “predicted operon” /note = “ordered genes contained in theoperon: b0318 b0319 b0320 b0321 b0322 ” lux-a.pk0026.g2 − Operoncomplement(786066 . . . 786818) /note = “predicted operon” /note =“ordered genes contained in the operon: gpmA ” lux-a.pk032.e4 + Operon(2141288 . . . 2144605) /note = “predicted operon” /note = “orderedgenes contained in the operon: yegE ” lux-a.pk037.c8 − Operoncomplement(262552 . . . 263231) /note = “predicted operon” /note =“ordered genes contained in the operon: b0245 b0246 ” lux-a.pk041.d8 −Operon complement(622777 . . . 623733) /note = “documented fepB operon”lux-a.pk047.c6 + Operon (3154754 . . . 3155464) /note = “predictedoperon” /note = “ordered genes contained in the operon: b3012 ”lux-a.pk052.f1 − Operon complement(3665421 . . . 3666818) /note =“predicted operon” /note = “ordered genes contained in the operon: yhjA” lux-a.pk058.f3 − Operon complement(3371333 . . . 3372124) /note =“predicted operon” /note = “ordered genes contained in the operon: b3226” lux-a.pk065.h2 − Operon complement(2009370 . . . 2009891) /note =“predicted operon” /note = “ordered genes contained in the operon: b1933b1934” lux-a.pk072.d1 − Operon complement(4300657 . . . 4301688) /note =“predicted operon” /note = “ordered genes contained in the operon: yjcR”lux-a.pk078.c2 − Operon complement(4474870 . . . 4475874) /note =“documented argI operon” lux-a.pk086.b7 + Operon (1481142 . . . 1484987)/note = “predicted operon” /note = “ordered genes contained in theoperon: hrpA ”

[0326] TABLE 19 lux-z.pk013.a15 − Operon complement(3366649 . . .3370239) note = “predicted operon” note = “ordered genes contained inthe operon: b3221 yhcI b3223 nanT” lux-z.pk013.g17 − Operoncomplement(4488774 . . . 4490093) note = “predicted operon” note =“ordered genes contained in the operon: yjgT” lux-z.pk013.i9 + Operon3128193 . . . 3129209 note = “predicted operon” note = “ordered genescontained in the operon: yi52_9” lux-z.pk013.k1 − Operoncomplement(2978786 . . . 2980204) note = “documented araE operon”lux-z.pk013.k11 + Operon 2027561 . . . 2028481 note = “predicted operon”note = “ordered genes contained in the operon: yedA” lux-z.pk013.k5 +Operon 2418641 . . . 2419288 note = “predicted operon” note = “orderedgenes contained in the operon: b2302” lux-z.pk013.o5 − Operoncomplement(1347004 . . . 1348209) note = “predicted operon” note =“ordered genes contained in the operon: b1287” lux-z.pk014.a14 + Operon1063259 . . . 1064515 note = “predicted operon” note = “ordered genescontained in the operon: yccE” lux-z.pk014.a22 + Operon 557402 . . .557977 note = “predicted operon” note = “ordered genes contained in theoperon: b0530” lux-z.pk014.c16 + Operon 607288 . . . 608400 note =“predicted operon” note = “ordered genes contained in the operon:yi81_2” lux-z.pk014.c5 − Operon complement(1561358 . . . 1565164) note =“predicted operon” note = “ordered genes contained in the operon: b1489b1490” lux-z.pk014.g11 − Operon complement(3055198 . . . 3056430) note =“documented serA operon” lux-z.pk014.h9 + Operon 948891 . . . 949481note = “predicted operon” note = “ordered genes contained in the operon:ycaK” lux-z.pk014.k13 + Operon 3769908 . . . 3773786 note = “documentedmtlADR operon” lux-z.pk014.l1 + Operon 3132146 . . . 3132838 note =“predicted operon” note = “ordered genes contained in the operon: b2986”lux-z.pk014.m1 + Operon 2752917 . . . 2753399 note = “predicted operon”note = “ordered genes contained in the operon: smpB” lux-z.pk014.n19 −Operon complement(4439115 . . . 4439753) note = “predicted operon” note= “ordered genes contained in the operon: msrA” lux-z.pk014.o9 − Operoncomplement(2486043 . . . 2488206) note = “predicted operon” note =“ordered genes contained in the operon: b2371 b2372” lux-z.pk014.p3 +Operon 4077307 . . . 4077549 note = “predicted operon” note = “orderedgenes contained in the operon: yiiF” lux-z.pk014.p4 − Operoncomplement(1930139 . . . 1932628) note = “predicted operon” note =“ordered genes contained in the operon: eda edd” lux-z.pk014.p8 − Operoncomplement 1122630 . . . 1123277) note = “predicted operon” note =“ordered genes contained in the operon: grxB” lux-z.pk015.a7 + Operon3408908 . . . 3409204 note = “documented fis operon” lux-z.pk015.b23 +Operon 4059826 . . . 4061091 note = “predicted operon” note = “orderedgenes contained in the operon: yihN” lux-z.pk015.c7 + Operon 2758568 . .. 2759194 note = “predicted operon” note = “ordered genes contained inthe operon: b2626” lux-z.pk015.d10 + Operon 2745907 . . . 2748081 note =“predicted operon” note = “ordered genes contained in the operon: b2611b2612 yfjD” lux-z.pk015.e17 − Operon complement(2794358 . . . 2794807)note = “predicted operon” note = “ordered genes contained in the operon:b2665” lux-z.pk015.f1 + Operon 2321467 . . . 2325313 note = “documentedatoDAB operon” lux-z.pk015.g1 + Operon 1509678 . . . 1515026 note =“predicted operon” note = “ordered genes contained in the operon: b1440b1441 b1442 b1443 b1444” lux-z.pk015.g13 + Operon 3214420 . . . 3215043note = “predicted operon” note = “ordered genes contained in the operon:b3071” lux-z.pk015.g23 − Operon complement(2828798 . . . 2830387) note =“predicted operon” note = “ordered genes contained in the operon: ygaA”lux-z.pk015.i5 − Operon complement(376759 . . . 377592) note =“predicted operon” note = “ordered genes contained in the operon: b0355”lux-z.pk015.n1 − Operon complement(586314 . . . 587204) note =“predicted operon” note = “ordered genes contained in the operon: ybcH”lux-z.pk015.n24 + Operon 2576686 . . . 2579659 note = “predicted operon”note = “ordered genes contained in the operon: b2464 tktB”lux-z.pk015.n4 − Operon complement(4145045 . . . 4145896) note =“predicted operon” note = “ordered genes contained in the operon: yijO”lux-z.pk015.p2 − Operon complement(3111560 . . . 3112492) note =“predicted operon” note = “ordered genes contained in the operon: b2972”lux-z.pk015.p6 + Operon 1768612 . . . 1768995 note = “predicted operon”note = “ordered genes contained in the operon: b1689” lux-z.pk016.a4 −Operon complement(4580819 . . . 4584385) note = “predicted operon” note= “ordered genes contained in the operon: hsdR” lux-z.pk016.c22 + Operon4210813 . . . 4211196 note = “predicted operon” note = “ordered genescontained in the operon: yjaA” lux-z.pk016.c9 + Operon 1073465 . . .1074103 note = “predicted operon” note = “ordered genes contained in theoperon: b1013” lux-z.pk016.e11 − Operon complement(2812905 . . .2814461) note = “predicted operon” note = “ordered genes contained inthe operon: gshA” lux-z.pk016.f16 + Operon 2405581 . . . 2406798 note =“predicted operon” note = “ordered genes contained in the operon: b2290”lux-z.pk016.f6 + Operon 1234161 . . . 1234880 note = “predicted operon”note = “ordered genes contained in the operon: fadR” lux-z.pk016.h16 +Operon 3054261 . . . 3054809 note = “predicted operon” note = “orderedgenes contained in the operon: ygfA” lux-z.pk016.i17 − Operoncomplement(2817403 . . . 2820033) note = “predicted operon” note =“ordered genes contained in the operon: alaS” lux-z.pk016.i2 + Operon4041737 . . . 4043209 note = “predicted operon” note = “ordered genescontained in the operon: yihF” lux-z.pk016.i24 + Operon 1055484 . . .1056512 note = “predicted operon” note = “ordered genes contained in theoperon: torT” lux-z.pk016.n14 − Operon complement(1228706 . . . 1229623)note = “predicted operon” note = “ordered genes contained in the operon:b1182” lux-z.pk016.o11 − Operon complement(4108320 . . . 4109087) note =“predicted operon” note = “ordered genes contained in the operon: tpiA”lux-z.pk016.o17 + Operon 2475867 . . . 2478550 note = “documented dsdXAoperon” lux-z.pk017.c2 + Operon 3550718 . . . 3553423 note = “documentedmalT operon” lux-z.pk017.g19 + Operon 420210 . . . 421583 note =“predicted operon” note = “ordered genes contained in the operon: b0402”lux-z.pk017.g23 − Operon complement(4468560 . . . 4469969) note =“documented pyrBI operon” lux-z.pk017.i21 − Operon complement(3633838 .. . 3635040) note = “predicted operon” note = “ordered genes containedin the operon: yhiN” lux-z.pk017.i4 + Operon 3004356 . . . 3005447 note= “predicted operon” note = “ordered genes contained in the operon:b2870” lux-z.pk017.k15 − Operon complement(2657583 . . . 2659575) note =“predicted operon” note = “ordered genes contained in the operon: yfhFb2529 b2530” lux-z.pk017.m18 + Operon 2270378 . . . 2275909 note =“predicted operon” note = “ordered genes contained in the operon: yejAyejB yejE yejF” lux-z.pk018.a16 − Operon complement(3738738 . . .3739211) note = “predicted operon” note = “ordered genes contained inthe operon: not-yiaI” lux-z.pk018.e4 + Operon 4256816 . . . 4257025 note= “predicted operon” note = “ordered genes contained in the operon:b4045” lux-z.pk018.g18 − Operon complement(2488276 . . . 2489970) note =“predicted operon” note = “ordered genes contained in the operon: b2373”lux-z.pk018.i2 + Operon 2037500 . . . 2039140 note = “predicted operon”note = “ordered genes contained in the operon: b1971 b1972”lux-z.pk018.o10 + Operon 959463 . . . 960251 note = “predicted operon”note = “ordered genes contained in the operon: b0909” lux-z.pk019.a18 −Operon complement(2242798 . . . 2244789) note = “documented cirA operon”lux-z.pk019.a24 − Operon complement(4559066 . . . 4560244) note =“predicted operon” note = “ordered genes contained in the operon: yjiJ”lux-z.pk019.b15 + Operon 4611194 . . . 4611829 note = “predicted operon”note = “ordered genes contained in the operon: yjjV” lux-z.pk019.b21 −Operon complement(3309474 . . . 3310819) note = “predicted operon” note= “ordered genes contained in the operon: truB rbfA” lux-z.pk019.c6 −Operon complement(4388035 . . . 4389048) note = “predicted operon” note= “ordered genes contained in the operon: yjeQ” lux-z.pk019.e12 + Operon4263361 . . . 4264440 note = “predicted operon” note = “ordered genescontained in the operon: alr” lux-z.pk019.e2 + Operon 1225823 . . .1226191 note = “predicted operon” note = “ordered genes contained in theoperon: b1177” lux-z.pk019.g1 − Operon complement(663325 . . . 668151)note = “predicted operon” note = “ordered genes contained in the operon:rlpA mrdB mrdA ybeA ybeB” lux-z.pk019.h5 − Operon complement(1555136 . .. 1561100) note = “predicted operon” note = “ordered genes contained inthe operon: b1483 b1484 b1485 b1486 b1487 b1488” lux-z.pk019.j11 +Operon 2662383 . . . 2663264 note = “predicted operon” note = “orderedgenes contained in the operon: b2534” lux-z.pk019.j15 + Operon 1498597 .. . 1500179 note = “documented tehAB operon” lux-z.pk019.o21 − Operoncomplement(3961980 . . . 3963245) note = “predicted operon” note =“ordered genes contained in the operon: rhlB” lux-z.pk020.a23 + Operon3572704 . . . 3573297 note = “predicted operon” note = “ordered genescontained in the operon: b3434” lux-z.pk020.c16 − Operoncomplement(3809518 . . . 3810192) note = “predicted operon” note =“ordered genes contained in the operon: radC” lux-z.pk020.c8 + Operon4292060 . . . 4293373 note = “predicted operon” note = “ordered genescontained in the operon: gltP” lux-z.pk020.e3 + Operon 167484 . . .173444 note = “documented fhuACDB operon” lux-z.pk020.h21 + Operon4007918 . . . 4008433 note = “predicted operon” note = “ordered genescontained in the operon: yigL” lux-z.pk020.j20 − Operoncomplement(379293 . . . 380511) note = “predicted operon” note =“ordered genes contained in the operon: b0358 b0359” lux-z.pk020.j9 +Operon 2322776 . . . 2324098 note = “predicted operon” note = “orderedgenes contained in the operon: atoE” lux-z.pk020.l21 + Operon 1329072 .. . 1331669 note = “predicted operon” note = “ordered genes contained inthe operon: topA” lux-z.pk020.n12 − Operon complement(216179 . . .218775) note = “predicted operon” note = “ordered genes contained in theoperon: yaeF proS” lux-z.pk021.a14 + Operon 4277559 . . . 4279208 note =“predicted operon” note = “ordered genes contained in the operon: yjcE”lux-z.pk021.b3 − Operon complement(2962383 . . . 2964059) note =“documented lgt-thyA operon” lux-z.pk021.c10 + Operon 3407917 . . .3408882 note = “predicted operon” note = “ordered genes contained in theoperon: yhdG” lux-z.pk021.d1 + Operon 1687876 . . . 1689384 note =“predicted operon” note = “ordered genes contained in the operon: b1614”lux-z.pk021.d22 − Operon complement(4523674 . . . 4525548) note =“predicted operon” note = “ordered genes contained in the operon: yjhJyjhK yjhL” lux-z.pk021.e10 − Operon complement(2287085 . . . 2288101)note = “predicted operon” note = “ordered genes contained in the operon:yi52_8” lux-z.pk021.g16 − Operon complement(2421756 . . . 2423936) note= “predicted operon” note = “ordered genes contained in the operon: hisPhisM hisQ” lux-z.pk021.h14 − Operon complement(3096577 . . . 3097584)note = “predicted operon” note = “ordered genes contained in the operon:yggM” lux-z.pk021.h3 + Operon 1805820 . . . 1806680 note = “predictedoperon” note = “ordered genes contained in the operon: b1725”lux-z.pk021.i24 + Operon 3814303 . . . 3815166 note = “predicted operon”note = “ordered genes contained in the operon: yicC” lux-z.pk021.k6 −Operon complement(4375389 . . . 4376522) note = “predicted operon” note= “ordered genes contained in the operon: ampC” lux-z.pk021.l19 − Operoncomplement(2660603 . . . 2661343) note = “predicted operon” note =“ordered genes contained in the operon: b2532” lux-z.pk021.n20 + Operon3225442 . . . 3228880 note = “predicted operon” note = “ordered genescontained in the operon: ygjJ ygjK” lux-z.pk021.n20 + Operon 3225442 . .. 3228880 note = “predicted operon” note = “ordered genes contained inthe operon: ygjj ygjK” lux-z.pk021.o16 − Operon complement(2710047 . . .2710904) note = “predicted operon” note = “ordered genes contained inthe operon: yfiC” lux-z.pk021.o4 + Operon 3490205 . . . 3491386 note =“predicted operon” note = “ordered genes contained in the operon: yhfC”lux-z.pk021.p19 + Operon 4199504 . . . 4200901 note = “documented hydHoperon” lux-z.pk022.b12 − Operon complement(635939 . . . 636841) note =“predicted operon” note = “ordered genes contained in the operon: b0603”lux-z.pk022.b20 − Operon complement(156299 . . . 156883) note =“predicted operon” note = “ordered genes contained in the operon: yadN”lux-z.pk022.c5 − Operon complement(2432102 . . . 2432761) note =“predicted operon” note = “ordered genes contained in the operon: dedA”lux-z.pk022.f12 + Operon 2264265 . . . 2265731 note = “predicted operon”note = “ordered genes contained in the operon: yeiQ” lux-z.pk022.g13 −Operon complement(2221958 . . . 2222899) note = “predicted operon” note= “ordered genes contained in the operon: pbpG” lux-z.pk022.g8 − Operoncomplement(3960360 . . . 3961844) note = “predicted operon” note =“ordered genes contained in the operon: gppA” lux-z.pk022.h21 + Operon2163690 . . . 2165051 note = “predicted operon” note = “ordered genescontained in the operon: b2081” lux-z.pk022.i1 − Operoncomplement(2940940 . . . 2941170) note = “predicted operon” note =“ordered genes contained in the operon: b2809” lux-z.pk022.j21 + Operon2597860 . . . 2598968 note = “predicted operon” note = “ordered genescontained in the operon: gcvR bcp” lux-z.pk022.k8 − Operoncomplement(4486129 . . . 4487631) note = “predicted operon” note =“ordered genes contained in the operon: yjgR” lux-z.pk022.n7 + Operon4002473 . . . 4003342 note = “predicted operon” note = “ordered genescontained in the operon: pldA” lux-z.pk022.p18 + Operon 1277180 . . .1278571 note = “documented narK operon” lux-z.pk023.a11 − Operoncomplement(4282992 . . . 4284950) note = “predicted operon” note =“ordered genes contained in the operon: acs” lux-z.pk023.c18 + Operon1194346 . . . 1195596 note = “documented icdA operon” lux-z.pk023.c21 +Operon 3387155 . . . 3388084 note = “predicted operon” note = “orderedgenes contained in the operon: b3243” lux-z.pk023.e17 + Operon 3418958 .. . 3420830 note = “predicted operon” note = “ordered genes contained inthe operon: yhdY yhdZ” lux-z.pk023.f12 + Operon 855186 . . . 856778 note= “predicted operon” note = “ordered genes contained in the operon:b0820” lux-z.pk023.g8 − Operon complement(2957082 . . . 2962199) note =“predicted operon” note = “ordered genes contained in the operon: recCppdC ygdB ppdB ppdA” lux-z.pk023.j2 − Operon complement(3436342 . . .3437146) note = “predicted operon” note = “ordered genes contained inthe operon: yhdM yhdN” lux-z.pk023.k11 − Operon complement(3239467 . . .3242381) note = “documented uxaCA operon” lux-z.pk023.k5 − Operoncomplement(1690914 . . . 1694095) note = “predicted operon” note =“ordered genes contained in the operon: uidB uidA” lux-z.pk023.l18 −Operon complement(1928481 . . . 1928771) note = “predicted operon” note= “ordered genes contained in the operon: yebG” lux-z.pk023.m21 + Operon3963846 . . . 3965291 note = “predicted operon” note = “ordered genescontained in the operon: rhoL rho” lux-z.pk023.m22 + Operon 1426547 . .. 1427008 note = “predicted operon” note = “ordered genes contained inthe operon: b1371” lux-z.pk023.m22 − Operon complement(3127058 . . .3128230) note = “predicted operon” note = “ordered genes contained inthe operon: b2981” lux-z.pk023.o7 + Operon 4380191 . . . 4381198 note =“predicted operon” note = “ordered genes contained in the operon: yjeA”lux-z.pk023.o8 + Operon 2263215 . . . 2264042 note = “predicted operon”note = “ordered genes contained in the operon: yeiP” lux-z.pk025.b18 −Operon complement(1186342 . . . 1187472) note = “predicted operon” note= “ordered genes contained in the operon: ycfD” lux-z.pk025.b8 − Operoncomplement(1349852 . . . 1355134) note = “predicted operon” note =“ordered genes contained in the operon: sapF sapD sapC sapB sapA”lux-z.pk025.c11 + Operon 1146017 . . . 1146538 note = “predicted operon”note = “ordered genes contained in the operon: yceD” lux-z.pk025.d13 −Operon complement(2549297 . . . 2550269) note = “predicted operon” note= “ordered genes contained in the operon: b2433 b2434” lux-z.pk025.e10 +Operon 1391230 . . . 1392864 note = “predicted operon” note = “orderedgenes contained in the operon: b1329” lux-z.pk025.f5 − Operoncomplement(4487709 . . . 4488707) note = “predicted operon” note =“ordered genes contained in the operon: yjgS” lux-z.pk025.h13 − Operoncomplement(1731778 . . . 1732125) note = “predicted operon” note =“ordered genes contained in the operon: ydhD” lux-z.pk025.h22 + Operon4098391 . . . 4099011 note = “documented sodA operon” lux-z.pk025.i24 −Operon complement(1261249 . . . 1262723) note = “predicted operon” note= “ordered genes contained in the operon: ychB hemM” lux-z.pk025.j11 −Operon complement(529356 . . . 530450) note = “predicted operon” note =“ordered genes contained in the operon: ybbB” lux-z.pk025.j16 + Operon1712401 . . . 1713006 note = “predicted operon” note = “ordered genescontained in the operon: gst” lux-z.pk025.j4 + Operon 3578769 . . .3S79257 note = “predicted operon” note = “ordered genes contained in theoperon: b3441” lux-z.pk025.m6 − Operon complement(1581786 . . . 1581983)note = “predicted operon” note = “ordered genes contained in the operon:b1500” lux-z.pk025.m7 + Operon 3775026 . . . 3776681 note = “documentedlldP operon” lux-z.pk025.o12 + Operon 3057773 . . . 3058666 note =“predicted operon” note = “ordered genes contained in the operon: iciA”lux-z.pk025.o8 + Operon 402927 . . . 404042 note = “predicted operon”note = “ordered genes contained in the operon: yaiC” lux-z.pk018.i18 −surA lux-z.pk013.g19 + yacK lux-z.pk014.h24 − map lux-z.pk014.l8 + betTlux-z.pk020.g15 + yaiB lux-z.pk017.o4 − proC lux-z.pk023.g7 − bioAlux-t.pk001.a1 osmY lux-t.pk001.a2 inaA lux-t.pk001.a3 hisP1lux-a.pk068.c1 hisP3 lux-t.pk001.a4 katG lux-t.pk001.a5 yebFlux-t.pk001.a6 flhB lux-t.pk001.a7 ppa lux-t.pk001.a8 sgcR(yjhJ)lux-t.pk001.a9 flhC lux-a.pk0022.d4 recA

[0327] TABLE 20 1st gene of the operon 45 minutes 90 minutes 135 minutesb0116 (lpdA) >2× >2× * b0168 (map) >2× >2× * b0767 (ybhE) >2× >2× >2×b0842 >2× >2× >2× b1186 (nhaB) >2× >2× >2× b1413 (hrpA) >2× >2× >2×b1676 (pykF) >2× >2× >2× b1993 (cobU) >2× >2× >2× b2081 >2× >2× *b2999 >2× * * b3012 >2× >2× >2× b3904 (rhaB) >2× >2× >2× b4106(phnC) >2× >2× >2× b4392 (slt) >2× >2× * b0314 (betT) * >2× >2×b0417 * >2× >2× b0422 (xseB) * >2× * b0572 * >2× >2× b0593(entC) * >2× >2× b0839 (dacC) * >2× >2× b1188 (ycgB) * >2× * b1223(narK) * >2× >2× b1872 (bisZ) * >2× >2× b2237 (inaA) * >2× >2×b2322 * >2× >2× b2367 (emrY) * >2× * b2451 * >2× >2× b2550 * >2× * b2699(recA) * >2× >2× b3245 * >2× >2× b3267 (yhdV) * >2× >2× b3336(bfr) * >2× * b3365 (nirB) * >2× >2× b3419 (yhgJ) * >2× >2× b3666(uhpT) * >2× >2× b3942 (katG) * >2× >2× b4043 (lexA) * >2× * b4264(yjgS) * >2× b0005 * >2× b0123 (yacK) * >2× b0386 (proC) * * >2× b0450(glnK) * * >2× b0774 (bioA) * * >2× b1847 (yebF) * * >2× b1852(zwf) * * >2× b2019 (hisG) * * >2× b2114 (metG) * * >2× b2428 * * >2×b3573 * * >2× b3779 (gppA) * * >2× b4226 (ppa) * * >2×

[0328]

1 4 1 20 DNA Artificial Sequence Description of Artificial Sequenceprimer 1 ggatcggaat tcccggggat 20 2 20 DNA Artificial SequenceDescription of Artificial Sequence primer 2 ctggccgtta ataatgaatg 20 325 DNA Artificial Sequence Description of Artificial Sequence primer 3ggaattgggg atcggagctc ccggg 25 4 25 DNA Artificial Sequence Descriptionof Artificial Sequence primer 4 gaatggcgcg aattcggtac ccggg 25

What is claimed is:
 1. A method for determining gene function between atleast two genome-registered collections comprising: (a) assembling atleast two genome-wide scale, genome-registered collections; (b)perturbing each collection from (a) with at least one perturbation; (c)measuring the response of each collection to each perturbation of (b);(d) analyzing the results of the at least one perturbation to identifypatterns of similarities and differences between the at least twogenome-registered collections.
 2. A method according to claim 1 whereinthe perturbation is selected from the group consisting of radiation,humidity, alterations in temperature, alterations in carbon source,alterations in energy source, alterations in nitrogen source,alterations in phosphorus source, alterations in sulfur source,alterations in trace element sources, a change in pH, the presence otherorganisms, the presence of chemicals, the presence of toxins, andabnormal levels of normal metabolites.
 3. A method for generating agenome-registered collection of reporter gene fusions comprising thesteps of: (a) generating a set of gene fusions comprising: 1) a reportergene or reporter gene complex operably linked to 2) a genomic fragmentfrom an organism of which at least 15% of the genomic nucleotidesequence is known; (b) introducing in vitro the reporter gene fusionsfrom step (a) into a host organism; (c) registering the reporter genefusions on the basis of sequence homology to the genomic sequence of theorganism; (d) repeating (a), (b), and/or (c) until reporter gene fusionshave been made to at least 15% of the known genomic nucleotide sequenceof said organism.
 4. A method according to claim 3 wherein the genefusions of step (a) are generated either in vivo or in vitro.
 5. Amethod for generating a genome-registered collection of reporter genefusions comprising: (a) generating random nucleic acid fragments fromthe DNA of an organism of which at least 15% of the nucleotide sequenceis known; (b) operably linking the random nucleic acid fragmentsgenerated in (a) to a vector containing a promoterless reporter gene orreporter gene complex; (c) introducing the vector (b) containing thegene fusions into a host organism; (d) determining the nucleic acidsequence of the distal and the proximal ends of the random nucleicfragments relative to the reporter gene or reporter gene complex; (e)registering the sequenced fusions of step (d) on the basis of sequencehomology to the genomic sequence of the host organism; (d) repeating(a), (b), and/or (c) until reporter gene fusions have been made to atleast 15% of the known genomic nucleotide sequence of said organism
 6. Amethod according to claim 5 wherein the random nucleic acid fragments ofstep (a) are generated by method selected from the group consisting ofrestriction enzyme digestion, physical shearing of the genome andpolymerase chain reaction.
 7. A method for generating agenome-registered collection of reporter gene fusions comprising: (a)providing a genome from an organism wherein at least 15% of thenucleotide sequence is known; (b) providing a series of amplificationprimers having homology to specific known regions of the genome of (a);(c) amplifying portions of the genome of (a) with the primers of (b) tocreate a collection of nucleic acid amplification products; (d) operablylinking the amplification products of (c) to a vector containing apromoterless reporter gene or reporter gene complex; (e) introducing thereporter gene fusions into a said organism; (f) repeating (a)-(e) until,until reporter gene fusions have been made to at least 15% of the knowngenomic nucleotide sequence of said organism.
 8. A method for generatinga genome-registered collection of reporter gene fusions comprising stepsof: (a) introducing one or more transposons into the genome of anorganism of which at least 15% of the nucleotide sequence is known, eachtransposon containing a promoterless reporter gene or reporter genecomplex; (b) determining the nucleic acid sequence of the junctionbetween the proximal end of the genomic DNA and the transposoncontaining the reporter gene or reporter gene complex and registeringthe reporter gene fusions relative to the genomic sequence of theorganism, (c) repeating (a) and (b) until reporter gene fusions havebeen made to at least 15% of the known genomic nucleotide sequence ofsaid organism
 9. A method according to any one of claims 1, 3, 5, 7 or 8wherein organism is selected from the group consisting of prokaryotesand fungi.
 10. A method according to claim 9 wherein the prokaryote isan enteric bacterium.
 11. A method according to claim 10 wherein theenteric bacterium is selected from the group consisting of Escherichiaand Salmonella.
 12. A method according to one of claims 1, 3, 5, 7 or 8wherein the reporter gene or reporter gene complex is selected from thegroup consisting of luxCDABE, lacZ, gfp, cat, galK, inaZ, luc, luxAB,bgaB, nptII, phoA, uidA and xylE.
 13. A method according to one ofclaims 1, 3, 5, 7 or 8 wherein at least 50% of the genomic nucleotidessequence is known.
 14. A method for identifying a profile of inducingconditions for a reporter gene fusion comprising: (a) obtaining a geneexpression profile of an organism under induced and non-inducedconditions wherein induced genes are identified; (b) providing agenome-registered collection of reporter gene fusions, said fusionsregistered to the genome of the organism of (a); (c) selecting thereporter gene fusions of (b) that correspond to the induced genes of (a)to create a subset of the genome-register collection; (d) contacting thesubset of the genome-register collection of (c) with the inducingconditions of (a) to identify at least one representative reporter genefusion whose expression was altered in a similar manner as in (a); (e)contacting the at least one representative reporter gene fusion of (d)in a high throughput manner with a multiplicity of different inducingconditions to identify a profile of inducing conditions for thatreporter gene fusion.
 15. A method according to claim 14 wherein atleast 15% of the genomic nucleotide sequence of said organism is known.16. A method for identifying a profile of inducing conditions for areporter gene fusion comprising: (a) obtaining a gene expression profilefor each of mutant strain and a parental strain organism under inducedand non-induced conditions wherein induced genes are identified; (b)providing a genome-registered collection of reporter gene fusions, saidfusions registered to the genome of the organism of (a); (c) selectingthe reporter gene fusions of (b) that correspond to the induced genes of(a) to create a subset of the genome-register collection; (d) contactingthe subset of the genome-register collection of (c) with the inducingconditions of (a) to identify at least one representative reporter genefusion whose expression was altered in a similar manner as in (a); (e)contacting the at least one representative reporter gene fusion of (d)in a high throughput manner with a multiplicity of different inducingconditions to identify a profile of inducing conditions for thatreporter gene fusion.
 17. A method to validate results fromcomprehensive genome analysis comprising the steps of: (a) analyzing agenome-wide, gene expression assay of an organism treated with acondition or chemical of interest to identify genes with alteredexpression; (b) selecting from a genome-registered collection ofreporter gene fusions those reporter gene fusions containing promoterregions operably linked to genes corresponding to the altered genes from(a) or genes co-regulated with genes corresponding to the altered genesfrom (a); (c) testing expression of the reporter gene fusions selectedfrom (b) with the conditions or chemicals of interest used in (a); and(d) comparing the gene expression results from (c) to the geneexpression result of (a).
 18. A method to determine operon structurecomprising steps of: (a) selecting a subset of reporter gene fusionsfrom a genome-registered collection of reporter gene fusions that map tothe region of a possible operon; (b) assaying the subset for thereporter gene function; and (c) determining a putative operon structurebased on the quantities of reporter gene function.
 19. A method forconstructing a cellular array containing reporter gene fusionscomprising: (a) generating a set of gene fusions comprising: 1) areporter gene or reporter gene complex operably linked to 2) a genomicfragment from an organism of which at least 15% of the genomicnucleotide sequence is known; (b) selecting a non-redundant subset ofreporter gene fusions from the set of (a) representative of at least 15%of known or suspected promoter regions from a genome-registeredcollection of reporter gene fusions, each containing a known orsuspected promoter region operably linked to a reporter gene or reportergene complex; and (c) fixing the non-redundant subset of reporter genefusions of (b) in an array format.
 20. A method for measuring geneexpression responses to perturbation comprising: (a) constructing atleast 2 identical cellular arrays, each cellular array comprising areporter gene fusion comprising: 1) a reporter gene or reporter genecomplex operably linked to 2) a genomic fragment from an organism ofwhich at least 15% of the genomic nucleotide sequence is known; whereinat least one cellular array is a control array and at least one cellulararray is an experimental array; (b) contacting the experimental array of(a) with a perturbing condition; (c) comparing the differences betweenthe gene expression activity of the control and the experimental arraywherein gene expression response to a perturbing condition isdetermined.
 21. The method of claim 20 wherein the cellular array isfixed in a manner selected from the group consisting of, fixed on asolid medium, and arrayed in liquid medium.
 22. The method of claim 20wherein the perturbing condition is selected from the group consistingof radiation, humidity, alterations in temperature, alterations incarbon source, alterations in energy source, alterations in nitrogensource, alterations in phosphorus source, alterations in sulfur source,alterations in trace element sources, a change in pH, the presence otherorganisms, the presence of chemicals, the presence of toxins, andabnormal levels of normal metabolites.